Companion diagnostic for anti-hyaluronan agent therapy and methods of use thereof

ABSTRACT

Methods and diagnostic agents for identification of subjects for cancer treatment with an anti-hyaluronan agent, such as a hyaluronan-degrading enzyme, are provided. Diagnostic agents for the detection and quantification of hyaluronan in a biological sample and monitoring cancer treatment with an anti-hyaluronan agent, for example a hyaluronan-degrading enzyme, are provided. Combinations and kits for use in practicing the methods also are provided.

RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 13/694,071, now allowed, entitled “Companion Diagnostic forAnti-Hyaluronan Agent Therapy and Methods of Use Thereof,” filed Oct.24, 2012, which claims priority to U.S. Provisional Application Ser. No.61/628,187, filed Oct. 24, 2011, entitled “Companion Diagnostic ForHyaluronan-Degrading Enzyme Therapy and Methods of Use Thereof,” to U.S.Provisional Application No. 61/559,011, filed Nov. 11, 2011, entitled“Companion Diagnostic for Hyaluronan-Degrading Enzyme Therapy andMethods of Use Thereof,” to U.S. Provisional Application No. 61/630,765,filed Dec. 16, 2011, entitled “Companion Diagnostic forHyaluronan-Degrading Enzyme Therapy and Methods of Use Thereof,” and toU.S. Provisional Application No. 61/714,700, filed Oct. 16, 2012,entitled “Companion Diagnostic for Anti-Hyaluronan Agent Therapy andMethods of Use Thereof. The subject matter of each of the above-notedapplications is incorporated by reference in its entirety.

This application is related to International PCT Patent Application No.PCT/US2012/061743, filed Oct. 24, 2012, entitled “Companion Diagnosticfor Anti-Hyaluronan Agent Therapy and Methods of Use Thereof,” whichclaims priority to U.S. Provisional Application Ser. Nos. 61/628,187;61/559,011; 61/630,765 and 61/714,700.

The subject matter of the above-noted related application isincorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

An electronic version of the Sequence Listing is filed herewith, thecontents of which are incorporated by reference in their entirety. Theelectronic file was created on Aug. 12, 2014, is 1,827 kilobytes insize, and is titled 3096Bseq001.txt.

FIELD OF INVENTION

Methods and diagnostic agents for identification of subjects for cancertreatment with a hyaluronan-degrading enzyme are provided. Diagnosticagents for the detection and quantification of hyaluronan in abiological sample and monitoring cancer treatment with ahyaluronan-degrading enzyme are provided. Combinations and kits for usein practicing the methods also are provided.

BACKGROUND

Hyaluronan-degrading enzymes have been used therapeutically, typicallyas dispersing and spreading agents in combination with other therapeuticagents. Hyaluronan-degrading enzymes also can be used in single-agenttherapy for the treatment of hyaluronan-associated diseases anddisorders. For example, tumors and cancers are associated withaccumulation of hyaluronan and treatment with a hyaluronan-degradingenzyme inhibits the growth of tumor and increases vascular perfusion andimproves delivery of chemotherapeutic agents to the tumor. There existsa need for methods and reagents for improving treatment of patients whoare treated with hyaluronan-degrading enzymes.

SUMMARY

Provided herein is a method for selecting a subject for treatment of atumor with an anti-hyaluronan agent, for example, a hyaluronan-degradingenzyme. In the provided method, a tissue or body fluid sample from asubject with a tumor or cancer is contacted with a hyaluronan bindingprotein (HABP) that has not been prepared from or isolated from animalcartilage. Binding of the hyaluronan binding protein to the sample isdetected, thereby determining the amount of hyaluronan in the sample,wherein if the amount of hyaluronan in the sample is at or above apredetermined threshold level, selecting the subject for treatment withan anti-hyaluronan agent, for example, a hyaluronan degrading enzyme.

Provided herein is a method for selecting a subject for treatment of atumor with an anti-hyaluronan agent, for example a hyaluronan-degradingenzyme, wherein a body fluid from a subject with a tumor or cancer iscontacted with a hyaluronan binding protein (HABP) that has not beenprepared from or isolated from animal cartilage and binding of the HABPto the sample is effected by a solid-phase binding assay with acolorimetric or fluorescent signal, thereby determining the amount ofhyaluronan in the sample, wherein a subject is selected for treatmentwith am anti-hyaluronan agent, for example a hyaluronan degradingenzyme, when the predetermined threshold level is high HA. In someexamples of the method, the predetermined threshold level is at least orabove 0.025 μg HA/ml of sample, 0.030 μg/ml, 0.035 μg/ml, 0.040 μg/ml,0.045 μg/ml, 0.050 μg/ml, 0.055 μg/ml, 0.060 μg/ml, 0.065 μg/ml, 0.070μg/ml, 0.08 μg/ml, 0.09 μg/ml. 0.1 μg/ml, 0.2 μg/ml, 0.3 μg/ml orhigher.

Provided herein is a method for selecting a subject for treatment of atumor with an anti-hyaluronan agent, for example a hyaluronan-degradingenzyme, wherein a tumor tissue sample from a subject with a tumor orcancer is contacted with a hyaluronan binding protein (HABP) that hasnot been prepared from or isolated from animal cartilage and binding ofthe HABP to the sample is effected by histochemistry, therebydetermining the amount of hyaluronan in the sample, wherein a subject isselected for treatment with an anti-hyaluronan agent, for example ahyaluronan degrading enzyme, when the predetermined threshold level isan HA score of at least +2 (HA⁺²) or at least +3 (HA⁺³). In someexamples, the predetermined threshold level is an HA score of at least+3 (HA⁺³) (high levels). In other examples, the predetermined thresholdlevel is at least a percent HA positive pixels in tumor (cells andstroma) to total stain in tumor tissue of at least 10%, 10% to 25% orgreater than 25%. For example, the predetermined threshold level is atleast a percent HA positive pixels in tumor (cells and stroma) to totalstain in tumor tissue of at least 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40%.

Also provided herein is a method for selecting a subject for treatmentof a tumor with an anti-hyaluronan agent, for example ahyaluronan-degrading enzyme, wherein the subject is then treated withthe anti-hyaluronan agent, for example a hyaluronan-degrading enzyme. Insome examples, the anti-hyaluronan agent is a hyaluronan-degradingenzyme that is administered in a dosage range amount of between or aboutbetween 0.01 μg/kg (of the subject) to 50 μg/kg, 0.01 μg/kg to 20 μg/kg,0.01 μg/kg to 15 μg/kg, 0.05 μg/kg to 10 μg/kg, 0.75 μg/kg to 7.5 μg/kgor 1.0 μg/kg to 3.0 μg/kg and a frequency of administration is twiceweekly, once weekly, once every 14 days, once every 21 days or onceevery month. In particular examples of the method, a corticosteroid isadministered prior to administration of a hyaluronan-degrading enzyme orafter administration of the hyaluronan-degrading enzyme, typically in anamount sufficient to ameliorate an adverse effect in the subject fromthe administered hyaluronan-degrading enzyme. For example, the amount ofcorticosteroid administered is between at or about 0.1 to 20 mgs, 0.1 to15 mgs, 0.1 to 10 mgs, 0.1 to 5 mgs, 0.2 to 20 mgs, 0.2 to 15 mgs, 0.2to 10 mgs, 0.2 to 5 mgs, 0.4 to 20 mgs, 0.4 to 15 mgs, 0.4 to 10 mgs,0.4 to 5 mgs, 0.4 to 4 mgs, 1 to 20 mgs, 1 to 15 mgs or 1 to 10 mgs.

Also provided herein is a method for predicting efficacy of treatment ofa subject with an anti-hyaluronan agent, for example a hyaluronandegrading enzyme. In the provided method, a tissue or body fluid samplefrom a subject who is or has been treated with an anti-hyaluronan agent,for example a hyaluronan degrading enzyme, is contacted with ahyaluronan binding protein (HABP) that has not been prepared from orisolated from animal cartilage and binding of the hyaluronan bindingprotein to the sample is detected, thereby determining the amount ofhyaluronan in the sample, wherein detection of a decrease in hyaluronancompared to before treatment with the anti-hyaluronan agent (e.g.hyaluronan-degrading enzyme) or the last dose of anti-hyaluronan agent(e.g. hyaluronan-degrading enzyme) indicates that the treatment iseffective.

Also provided herein is a method for monitoring treatment of a subjectwith an anti-hyaluronan agent (e.g. a hyaluronan degrading enzyme). Inthe provided method, a tissue or body fluid sample from a subject with atumor or cancer is contacted with a hyaluronan binding protein (HABP)that has not been prepared from or isolated from animal cartilage andthe amount of hyaluronan binding protein that binds to the sample isdetected, thereby determining the amount of hyaluronan in the sample,and the level of hyaluronan is compared to a control or reference sampleto thereby determine the amount of hyaluronan in the sample relative tothe control or reference sample, wherein the amount of hyaluronan is anindicator of the progress of treatment.

Also provided herein are methods for predicting efficacy of treatment ofa subject with an anti-hyaluronan agent (e.g. a hyaluronan degradingenzyme) and monitoring treatment of a subject with an anti-hyaluronanagent (e.g. a hyaluronan degrading enzyme) wherein treatment is alteredbased on the determined amount of hyaluronan in the sample relative tothe control or reference sample, such that if the amount of hyaluronanin the sample is at or above the amount in the control or referencesample, treatment is continued or escalated by increasing the dosageand/or dose schedule; or if the amount of hyaluronan in the sample isbelow the amount in the control or reference sample, treatment iscontinued, reduced by decreasing the dosage and/or dose schedule, orterminated. In some examples, the control or reference sample is asample from a healthy subject, is a baseline sample from the subjectprior to treatment with an anti-hyaluronan agent (e.g.hyaluronan-degrading enzyme) or is a sample from a subject prior to thelast dose of anti-hyaluronan agent (e.g. hyaluronan-degrading enzyme).In some examples, the subject has a tumor or cancer and the sample is atumor tissue sample and detection is effected by histochemistry. Inother examples, the subject has a tumor or cancer and the sample is abody fluid and detection is effected by a solid-phase binding assay. Insome examples, the solid-phase binding assay is a microtiter plate assayand binding is detected colorimetrically or via fluorescence.

In any of the methods provided herein, the step of contacting the samplewith an HABP can be effected at between or about between pH 5.6 to 6.4.For example, the step of contacting the sample with an HABP is effectedat a pH of about 5.8, 5.9, 6.0, 6.1 or 6.2. In some examples, the HABPspecifically binds to HA with a binding affinity represented by thedissociation constant (Kd) of at least less than or less than or 1×10⁻⁷M, 9×10⁻⁸ M, 8×10⁻⁸ M, 7×10⁻⁸ M, 6×10⁻⁸ M, 5×10⁻⁸ M, 4×10⁻⁸ M, 3×10⁻⁸ M,2×10⁻⁸ M, 1×10⁻⁸ M, 9×10⁻⁹ M, 8×10⁻⁹ M, 7×10⁻⁹ M, 6×10⁻⁹ M, 5×10⁻⁹ M,4×10⁻⁹ M, 3×10⁻⁹ M, 2×10⁻⁹ M, 1×10⁻⁹ M or lower Kd.

In any of the methods provided herein, the HABP can be generatedrecombinantly or synthetically. In some examples, the HABP contains alink module. In other examples, the HABP contains two or more linkmodules. In further examples, the link module or modules are the onlyHABP portion of the molecule. Thus, provided herein are methods whereinthe HABP contains a link module selected from among CD44, LYVE-1,HAPLN1/link protein, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican,neurocan, brevican, phosphacan, TSG-6, Stabilin-1, Stabilin-2, CAB61358and KIAA0527. In some examples of the method, the HABP contains aportion of a CD44, LYVE-1, HAPLN1/link protein, HAPLN2, HAPLN3, HAPLN4,aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, Stabilin-1,Stabilin-2, CAB61358 or KIAA0527 including a link module or a sufficientportion of a link module to bind HA. In a particular example, the HABPis a tumor necrosis factor-stimulated Gene (TSG-6) protein or a linkmodule of TSG-6 or a sufficient portion of a link module of TSG-6 tobind HA. In other examples of the method herein, the HABP contains a G1domain of a type C hyaluronan binding protein, for example, a G1 domainselected from among Aggrecan G1, Versican G1, Neurocan G1 and BrevicanG1. In particular examples, the G1 domain is the only HABP portion ofthe molecule.

In some examples of the methods provided herein, the HABP contains thesequence of amino acids set forth in any of SEQ ID NOS: 207, 222, 360,361, 371-394, 416-418 and 423-426 or has a sequence of amino acids thatexhibits at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%,96%, 97%, 98%, 99% or more sequence identity to a sequence of aminoacids set forth in any of SEQ ID NOS: 207, 222, 360, 361, 371-394,416-418 and 423-426 and specifically binds HA, or is an HA-bindingdomain thereof or a sufficient portion thereof to specifically bind toHA. In an exemplary example, the HABP contains a TSG-6 link module (LM)or a sufficient portion thereof that specifically binds HA. For example,the TSG-6-LM contains the sequence of amino acids set forth in SEQ IDNO: 207, 360, 417 or 418, or a sequence of amino acids comprising atleast 65% amino acid sequence identity to the sequence of amino acidsset forth in SEQ ID NO: 207, 360, 417 or 418 and specifically binds HA.In specific examples of the method, the HABP contains a link module setforth in SEQ ID NO:207 or a sequence of amino acids comprising at least65% amino acid sequence identity to the sequence of amino acids setforth in SEQ ID NO:207 and specifically binds HA. In other examples, theHABP contains a link module that exhibits at least 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity tothe sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or418, whereby the HABP specifically binds HA.

In some examples of the method provided herein, the TSG-6 link module ismodified to reduce or eliminate binding to heparin. For example, bindingto heparin is reduced at least 1.2-fold, 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, 100-fold or more. In some examples of themethod provided herein, TSG-6 link module contains an amino acidreplacement at an amino acid position corresponding to amino acidresidue 20, 34, 41, 54, 56, 72 or 84 set forth in SEQ ID NO:360, wherebya corresponding amino acid residue is identified by alignment to aTSG-6-LM set forth in SEQ ID NO:360. For example, the amino acidreplacement is in a TSG-6-LM set forth in SEQ ID NO:207 and the aminoacid replacement or replacements is at amino acid residue 21, 35, 42,55, 57, 73 or 85. The amino acid replacement can be to a non-basic aminoacid residue selected from among Asp (D), Glu (E), Ser (S), Thr (T), Asn(N), Gln (Q), Ala (A), Val (V), Ile (I), Leu (L), Met (M), Phe (F), Tyr(Y) and Trp (W). In a further example, the TSG-6 link module contains anamino acid replacement corresponding to amino acid replacement K20A,K34A or K41A in a TSG-6-LM set forth in SEQ ID NO:360 or the replacementat the corresponding residue in another TSG-6-LM. In another example,the TSG-6 link module contains amino acid replacements corresponding toamino acid replacements K20A, K34A and K41A in a TSG-6-LM set forth inSEQ ID NO:360 or the replacement at the corresponding residue in anotherTSG-6-LM. For example, the HABP contains a link module set forth in SEQID NO:361 or 416 or a sequence of amino acids comprising at least 65%amino acid sequence identity to the sequence of amino acids set forth inSEQ ID NO:361 or 416 that specifically binds HA.

In some examples of the methods provided herein, the HABP contains alink module that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequenceof amino acids set forth in SEQ ID NO:361 or 416, whereby the HABPspecifically binds HA. In particular examples, the HABP contains a linkmodule set forth in SEQ ID NO:361 or SEQ ID NO:416. In other examples,the link module is the only TSG-6 portion of the HABP. In some examplesof the methods provided herein, the HABP is a multimer containing afirst HA-binding domain linked directly or indirectly via a linker to amultimerization domain and a second HA-binding domain linked directly orindirectly via a linker to a multimerization domain. For example, theHA-binding domain is a link module or a G1 domain. The first and secondHA-binding domain can be the same or different. In a particular example,the first and second HA-binding domain is a TSG-6 link module, a variantthereof or a sufficient portion thereof that specifically binds to HA.For example, the TSG-6-LM contains a sequence of amino acids set forthin SEQ ID NO: 207, 360, 361, 416, 417 or 418 or a sequence of aminoacids comprising at least 65% amino acid sequence identity to thesequence of amino acids set forth in SEQ ID NO: 207, 360, 361, 416, 417or 418 that specifically binds HA. For example, the link module exhibitsat least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to the sequence of amino acids set forthin SEQ ID NO: 207, 360, 361, 416, 417 or 418, whereby the HABPspecifically binds HA. In some methods provided herein, the link modulecontains a sequence of amino acids set forth in SEQ ID NO: 207, 360,361, 416, 417 or 418.

In the provided methods, the HABPs, HA-binding domains, link modules orportions thereof can be linked to a multimerization domain that isselected from among an immunoglobulin constant region (Fc), a leucinezipper, complementary hydrophobic regions, complementary hydrophilicregions, compatible protein-protein interaction domains, free thiolsthat form an intermolecular disulfide bond between two molecules, and aprotuberance-into-cavity and a compensatory cavity of identical orsimilar size that form stable multimers. In a particular example, themultimerization domain is an Fc domain or a variant thereof that effectsmultimerization. For example, the Fc domain is from an IgG, IgM or anIgE, or the Fc domain has a sequence of amino acids set forth in SEQ IDNO:359. In some instances of the methods provided herein, the HABP is afusion protein that contains a TSG-6 link module and an immunoglobulinFc domain. For example, the HABP is TSG-6-LM-Fc that has a sequence ofamino acids set forth in SEQ ID NO: 212 or a sequence of amino acidsthat exhibits at least 65% amino acid sequence identity to SEQ ID NO:212and specifically binds HA, such as a sequence of amino acids thatexhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to the sequence of amino acids setforth in SEQ ID NO:212, whereby the HABP specifically binds HA. Inparticular examples, the HABP has a sequence of amino acids set forth inSEQ ID NO:212 or 215. In any of the methods provided herein the HABP canbe TSG-6-LM-Fc/ΔHep that has a sequence of amino acids set forth in SEQID NO: 215 or a sequence of amino acids that exhibits at least 65% aminoacid sequence identity to SEQ ID NO:215 and specifically binds HA, suchas a sequence of amino acids that exhibits at least 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity tothe sequence of amino acids set forth in SEQ ID NO:215, whereby the HABPspecifically binds HA.

In particular methods provided herein, the HABP is a TSG-6 orhyaluronan-binding region thereof. In some examples of the methodsprovided herein, the HABP or TSG-6 has a binding affinity to HA of atleast 1×10⁸ M⁻¹, 2×10⁸ M⁻¹, 3×10⁸ M⁻¹, 4×10⁸ M⁻¹, 5×10⁸ M⁻¹, 6×10⁸ M⁻¹,7×10⁸ M⁻¹, 8×10⁸ M⁻¹, 9×10⁸ M⁻¹, 1×10⁹ M⁻¹ or higher. In other examples,the HABP or TSG-6 is conjugated to a detectable moiety that isdetectably labeled or that can be detected. For example, the HABP orTSG-6 is biotinylated.

In some examples of the methods provided herein the sample is a stromaltissue sample, such as a stromal tissue sample from a tumor. The tissuesampled in the methods herein can be fixed, paraffin-embedded, fresh, orfrozen. In some examples, the sample is taken from a biopsy from a solidtumor, for example, obtained by needle biopsy, CT-guided needle biopsy,aspiration biopsy, endoscopic biopsy, bronchoscopic biopsy, bronchiallavage, incisional biopsy, excisional biopsy, punch biopsy, shavebiopsy, skin biopsy, bone marrow biopsy, and the Loop ElectrosurgicalExcision Procedure (LEEP). In other examples, the sample is a fluidsample that is a blood, serum, urine, sweat, semen, saliva, cerebralspinal fluid, or lymph sample. In any of the methods provided herein thesample can be obtained from a mammal. In an exemplary example, themammal is a human.

In any of the methods provided herein, the tumor can be of a cancerselected from among breast cancer, pancreatic cancer, ovarian cancer,colon cancer, lung cancer, non-small cell lung cancer, in situ carcinoma(ISC), squamous cell carcinoma (SCC), thyroid cancer, cervical cancer,uterine cancer, prostate cancer, testicular cancer, brain cancer,bladder cancer, stomach cancer, hepatoma, melanoma, glioma,retinoblastoma, mesothelioma, myeloma, lymphoma, and leukemia. In someexamples, the tumor is of a cancer that is a late-stage cancer, ametastatic cancer and an undifferentiated cancer.

In any of the methods provided herein, the anti-hyaluronan agent can bean agent that degrades hyaluronan or can be an agent that inhibits thesynthesis of hyaluronan. For example, the anti-hyaluronan agent can be ahyaluronan degrading enzyme. In another example, the anti-hyaluronanagent or is an agent that inhibits hyaluronan synthesis. For example,the anti-hyaluronan agent is an agent that inhibits hyaluronan synthesissuch as a sense or antisense nucleic acid molecule against an HAsynthase or is a small molecule drug. For example, an anti-hyaluronanagent is 4-methylumbelliferone (MU) or a derivative thereof, orleflunomide or a derivative thereof. Such derivatives include, forexample, a derivative of 4-methylumbelliferone (MU) that is6,7-dihydroxy-4-methyl coumarin or 5,7-dihydroxy-4-methyl coumarin.

In further examples of the methods provided herein, the hyaluronandegrading enzyme is a hyaluronidase. In some examples, thehyaluronan-degrading enzyme is a PH20 hyaluronidase or truncated formthereof to lacking a C-terminal glycosylphosphatidylinositol (GPI)attachment site or a portion of the GPI attachment site. In specificexamples, the hyaluronidase is a PH20 selected from a human, monkey,bovine, ovine, rat, mouse or guinea pig PH20. For example, thehyaluronan-degrading enzyme is a human PH20 hyaluronidase that isneutral active and N-glycosylated and is selected from among (a) ahyaluronidase polypeptide that is a full-length PH20 or is a C-terminaltruncated form of the PH20, wherein the truncated form includes at leastamino acid residues 36-464 of SEQ ID NO:1, such as 36-481, 36-482,36-483, where the full-length PH20 has the sequence of amino acids setforth in SEQ ID NO:2; or (b) a hyaluronidase polypeptide comprising asequence of amino acids having at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith the polypeptide or truncated form of sequence of amino acids setforth in SEQ ID NO:2; or (c) a hyaluronidase polypeptide of (a) or (b)comprising amino acid substitutions, whereby the hyaluronidasepolypeptide has a sequence of amino acids having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with the polypeptide set forth in SEQ ID NO:2 or thewith the corresponding truncated forms thereof. In exemplary examples,the hyaluronan-degrading enzyme is a PH20 that comprises a compositiondesignated rHuPH20.

In other examples, the anti-hyaluronan agent is a hyaluronan degradingenzyme that is modified by conjugation to a polymer. The polymer can bea PEG and the anti-hyaluronan agent a PEGylated hyaluronan degradingenzyme. Hence, in some examples of the methods provided herein thehyaluronan-degrading enzyme is modified by conjugation to a polymer. Forexample, the hyaluronan-degrading enzyme is conjugated to a PEG, thusthe hyaluronan degrading enzyme is PEGylated. In an exemplary example,the hyaluronan-degrading enzyme is a PEGylated PH20 enzyme (PEGPH20). Inthe methods provided herein, the corticosteroid can be a glucocorticoidthat is selected from among cortisones, dexamethasones, hydrocortisones,methylprednisolones, prednisolones and prednisones.

Also provided herein is a kit containing a hyaluronan binding protein(HABP) for detecting the amount of hyaluronan in a sample, wherein theHABP has not been prepared from animal cartilage and ahyaluronan-degrading enzyme. The HABP can be generated recombinantly orsynthetically. In some examples, the HABP contains one link module. Inother examples, the HABP contains two or more link modules. In someexamples, the link module or modules are the only HABP portion of themolecule. For example, the HABP contains a link module selected fromamong CD44, LYVE-1, HAPLN1/link protein, HAPLN2, HAPLN3, HAPLN4,aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, Stabilin-1,Stabilin-2, CAB61358 and KIA0527 or a portion thereof comprising a linkmodule or a sufficient portion of a link module to bind HA. In otherexamples, the HABP contains a G1 domain of a type C hyaluronan bindingprotein, for example, a G1 domain selected from among Aggrecan G1,Versican G1, Neurocan G1 and Brevican G1. In particular examples, the G1domain is the only HABP portion of the molecule.

In some examples, the kit contains an HABP containing the sequence ofamino acids set forth in any of SEQ ID NOS: 207, 222, 360, 361, 371-394and 416-418, and 423-426 or a sequence of amino acids that exhibits atleast 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%,98%, 99% or more sequence identity to a sequence of amino acids setforth in any of SEQ ID NOS: 207, 222, 360, 361, 371-394, 416-418 and423-426 and specifically binds HA, or an HA-binding domain thereof or asufficient portion thereof to specifically bind to HA. In an exemplaryexample, the HABP contains a TSG-6 link module (LM) or a sufficientportion thereof that specifically binds HA. For example, the TSG-6-LMcontains the sequence of amino acids set forth in SEQ ID NO: 207, 360,417 or 418, or a sequence of amino acids comprising at least 65% aminoacid sequence identity to the sequence of amino acids set forth in SEQID NO: 207, 360, 417 or 418 and specifically binds HA. In some examples,the HABP contains a link module that exhibits at least 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the sequence of amino acids set forth in SEQ ID NO:360,whereby the HABP binds HA. In particular examples, the HABP contains alink module set forth in SEQ ID NO: 207, 360, 417 or 418.

In some examples of the kits provided herein, the TSG-6 link module ismodified to reduce or eliminate binding to heparin. For example, bindingto heparin is reduced at least 1.2-fold, 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, 100-fold or more. In some examples, TSG-6link module contains an amino acid replacement at an amino acid positioncorresponding to amino acid residue 20, 34, 41, 54, 56, 72 or 84 setforth in SEQ ID NO:360, whereby a corresponding amino acid residue isidentified by alignment to a TSG-6-LM set forth in SEQ ID NO:360. Forexample, the amino acid replacement is to a non-basic amino acid residueselected from among Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln(Q), Ala (A), Val (V), Ile (I), Leu (L), Met (M), Phe (F), Tyr (Y) andTrp (W). Thus, provided herein is a kit wherein the TSG-6 link modulecontains an amino acid replacement corresponding to amino acidreplacement K20A, K34A or K41A in a TSG-6-LM set forth in SEQ ID NO:360or the replacement at the corresponding residue in another TSG-6-LM. Forexample, the TSG-6 link module contains amino acid replacementscorresponding to amino acid replacements K20A, K34A and K41A in aTSG-6-LM set forth in SEQ ID NO:360 or the replacement at thecorresponding residue in another TSG-6-LM. Also provided herein, arekits wherein the HABP contains a link module set forth in SEQ ID NO:361or 416 or a sequence of amino acids comprising at least 65% amino acidsequence identity to the sequence of amino acids set forth in SEQ IDNO:361 or 416 that specifically binds HA. For example, the HABP containsa link module that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequenceof amino acids set forth in SEQ ID NO:361 or 416, whereby the HABPspecifically binds HA. In particular examples, the HABP contains a linkmodule set forth in SEQ ID NO:361 or 416. In other examples, the linkmodule is the only TSG-6 portion of the HABP.

In some examples of the kits provided herein, the HABP is a multimercontaining a first HA-binding domain linked directly or indirectly via alinker to a multimerization domain and a second HA-binding domain linkeddirectly or indirectly via a linker to a multimerization domain. Forexample, the HA-binding domain is a link module or a G1 domain. Thefirst and second HA-binding domain can be the same or different. In aparticular example, the first and second HA-binding domain is a TSG-6link module, a variant thereof or a sufficient portion thereof thatspecifically binds to HA. For example, the TSG-6-LM contains a sequenceof amino acids set forth in SEQ ID NO: 207, 360, 361, 416, 417 or 418 ora sequence of amino acids comprising at least 65% amino acid sequenceidentity to the sequence of amino acids set forth in SEQ ID NO: 207,360, 361, 416, 417 or 418 that specifically binds HA. For example, thelink module exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence ofamino acids set forth in SEQ ID NO: 207, 360, 361, 416, 417 or 418,whereby the HABP specifically binds HA. In some methods provided herein,the link module contains a sequence of amino acids set forth in SEQ IDNO: 207, 360, 361, 416, 417 or 418.

In the provided kits, the HABPs can be linked by a multimerizationdomain that is selected from among an immunoglobulin constant region(Fc), a leucine zipper, complementary hydrophobic regions, complementaryhydrophilic regions, compatible protein-protein interaction domains,free thiols that form an intermolecular disulfide bond between twomolecules, and a protuberance-into-cavity and a compensatory cavity ofidentical or similar size that form stable multimers. In a particularexample, the multimerization domain is an Fc domain or a variant thereofthat effects multimerization. For example, the Fc domain is from an IgG,IgM or an IgE, or the Fc domain has a sequence of amino acids set forthin SEQ ID NO:359. In some instances of the methods provided herein, theHABP is a fusion protein that contains a TSG-6 link module and animmunoglobulin Fc domain. For example, the HABP is TSG-6-LM-Fc that hasa sequence of amino acids set forth in SEQ ID NO:212 or 215 or asequence of amino acids that exhibits at least 65% amino acid sequenceidentity to SEQ ID NO:212 or 215 and specifically binds HA, such as asequence of amino acids that exhibits at least 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to thesequence of amino acids set forth in SEQ ID NO:212 or 215, whereby theHABP specifically binds HA. In particular examples, the HABP has asequence of amino acids set forth in SEQ ID NO:212 or 215. In any of themethods provided herein the HABP can be TSG-6-LM-Fc/ΔHep that has asequence of amino acids set forth in SEQ ID NO: 215 or a sequence ofamino acids that exhibits at least 65% amino acid sequence identity toSEQ ID NO:215 and specifically binds HA, such as a sequence of aminoacids that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence ofamino acids set forth in SEQ ID NO:215, whereby the HABP specificallybinds HA.

In particular examples of kits provided herein, the HABP is a TSG-6 orhyaluronan-binding region thereof. In some examples of the methodsprovided herein, the HABP has a binding affinity, represented by theassociation constant (Ka), to HA of at least 10⁷ M⁻¹, for example, atleast 1×10⁸ M⁻¹, 2×10⁸ M⁻¹, 3×10⁸ M⁻¹, 4×10⁸ M⁻¹, 5×10⁸ M⁻¹, 6×10⁸ M⁻¹,7×10⁸ M⁻¹, 8×10⁸ M⁻¹, 9×10⁸ M⁻¹, 1×10⁹ M⁻¹ or higher. For example, theHABP has a binding affinity, represented by the dissociation constant(Kd), to HA of at least less than or less than 1×10⁻⁷ M, 9×10⁻⁸ M,8×10⁻⁸ M, 7×10⁻⁸ M, 6×10⁻⁸ M, 5×10⁻⁸ M, 4×10⁻⁸ M, 3×10⁻⁸ M, 2×10⁻⁸ M,1×10⁻⁸ M, 9×10⁻⁹ M, 8×10⁻⁹ M, 7×10⁻⁹ M, 6×10⁻⁹ M, 5×10⁻⁹ M, 4×10⁻⁹ M,3×10⁻⁹ M, 2×10⁻⁹ M, 1×10⁻⁹ M or lower Kd. In other examples, the HABP isconjugated to a detectable moiety that is detectably labeled or that canbe detected. For example, the HABP is biotinylated.

Also provided herein are kits containing a hyaluronan binding protein(HABP) for detecting the amount of hyaluronan in a sample, wherein theHABP has not been prepared from animal cartilage and an anti-hyaluronanagent (e.g. a hyaluronan-degrading enzyme). Any of the kits providedherein can further contain reagents for detection of the HABP. In anyexample of the kits provided herein, the anti-hyaluronan agent can beany described above or elsewhere herein. For example, theanti-hyaluronan agent can be a hyaluronan degrading enzyme such as ahyaluronidase. For example, the hyaluronan-degrading enzyme is a PH20hyaluronidase or truncated form thereof lacking a C-terminalglycosylphosphatidylinositol (GPI) attachment site or a portion of theGPI attachment site. In some examples, the PH20 is selected from ahuman, monkey, bovine, ovine, rat, mouse or guinea pig PH20. Forexample, the hyaluronan-degrading enzyme is a human PH20 hyaluronidasethat is neutral active and N-glycosylated and is selected from among (a)a hyaluronidase polypeptide that is a full-length PH20 or is aC-terminal truncated form of the PH20, wherein the truncated formincludes at least amino acid residues 36-464 of SEQ ID NO:1, wherein thefull-length PH20 comprises the sequence of amino acids set forth in SEQID NO:2; or (b) a hyaluronidase polypeptide comprising a sequence ofamino acids having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with thepolypeptide or truncated form of sequence of amino acids set forth inSEQ ID NO:2; or (c) a hyaluronidase polypeptide of (a) or (b) comprisingamino acid substitutions, whereby the hyaluronidase polypeptide has asequence of amino acids having at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith the polypeptide set forth in SEQ ID NO:2 or the with thecorresponding truncated forms thereof. In particular examples, thehyaluronan-degrading enzyme is a PH20 that comprises a compositiondesignated rHuPH20. In some examples, the hyaluronan-degrading enzyme ismodified by conjugation to a polymer, such as, for example, a PEG, andthe hyaluronan degrading enzyme is PEGylated. Therefore provided hereinis a kit wherein the hyaluronan-degrading enzyme is a PEGylated PH20enzyme (PEGPH20). Also provided herein are kits that further contain acorticosteroid. Any of the kits provided herein can further contain alabel or package insert for use of its components.

Provided herein are methods of use of a hyaluronan binding protein(HABP) for selecting a subject for treatment of a tumor with ananti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme), wherein theHABP has not been prepared or isolated from animal cartilage. Alsoprovided herein are pharmaceutical compositions containing a hyaluronanbinding protein (HABP) for use in selecting a subject for treatment of atumor with an anti-hyaluronan agent (e.g. a hyaluronan-degradingenzyme), wherein the HABP has not been prepared or isolated from animalcartilage.

Provided herein are methods of use of a hyaluronan binding protein(HABP) for predicting efficacy of treatment of a subject with ananti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme), wherein theHABP has not been prepared or isolated from animal cartilage. Alsoprovided herein are pharmaceutical compositions containing a hyaluronanbinding protein (HABP) for predicting efficacy of treatment of a subjectwith an anti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme),wherein the HABP has not been prepared or isolated from animalcartilage.

In any of the uses or pharmaceutical compositions provided herein, theHABP can contain a link module or modules or a G1 domain. In someexamples, the HABP contains a TSG-6 link module (LM), a variant thereofor a sufficient portion thereof that binds HA. In a particular example,the TSG-6 link module is modified to reduce or eliminate binding toheparin. In some examples, the HABP contains a sequence of amino acidsset forth in any of SEQ ID NOS: 207, 212, 215, 222, 360, 361, 371-394and 416-418, and 423-426 or a sequence of amino acids that exhibits atleast 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%,98%, 99% or more sequence identity to a sequence of amino acids setforth in any of SEQ ID NOS: 207, 212, 215, 222, 360, 361, 371-394 and416-418, and 423-426 and specifically binds HA, or an HA-binding domainthereof or a sufficient portion thereof to specifically bind to HA.

Also provided herein is a TSG-6-LM multimer containing a first linkmodule linked directly or indirectly via a linker to a multimerizationdomain and a second link module linked directly or indirectly via alinker to a multimerization domain, wherein the first and secondpolypeptide do not comprise the full-length sequence of TSG-6. In someexamples, the link module is the only TSG-6 portion of the firstpolypeptide and the second polypeptide. The first and second link modulecan be the same or different. In some examples, the link module containsa sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 418or a sequence of amino acids comprising at least 65% amino acid sequenceidentity to the sequence of amino acids set forth in SEQ ID NO: 207,360, 417 or 418 that specifically binds HA. For example, the link moduleexhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to the sequence of amino acids setforth in SEQ ID NO: 207, 360, 417 or 418 that specifically binds HA. Insome examples, the link module contains a sequence of amino acids setforth in SEQ ID NO: 207, 360, 417 or 418.

In some examples, the TSG-6 link module is modified to reduce oreliminate binding to heparin. Binding to heparin can be reduced at least1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold ormore. In some examples, the TSG-6 link module contains an amino acidreplacement at an amino acid position corresponding to amino acidresidue 20, 34, 41, 54, 56, 72 or 84 set forth in SEQ ID NO:360, wherebya corresponding amino acid residue is identified by alignment to aTSG-6-LM set forth in SEQ ID NO:360. For example, the amino acidreplacement is in a TSG-6-LM set forth in SEQ ID NO:207 and the aminoacid replacement or replacements is at amino acid residue 21, 35, 42,55, 57, 73 or 85. In some examples, the amino acid replacement is to anon-basic amino acid residue selected from among Asp (D), Glu (E), Ser(S), Thr (T), Asn (N), Gln (Q), Ala (A), Val (V), Ile (I), Leu (L), Met(M), Phe (F), Tyr (Y) and Trp (W). For example, the TSG-6 link modulecontains an amino acid replacement corresponding to amino acidreplacement K20A, K34A or K41A in a TSG-6-LM set forth in SEQ ID NO:360or the replacement at the corresponding residue in another TSG-6-LM. Ina particular example, the TSG-6 link module contains amino acidreplacements corresponding to amino acid replacements K20A, K34A andK41A in a TSG-6-LM set forth in SEQ ID NO:360 or the replacement at thecorresponding residue in another TSG-6-LM. In some examples, the linkmodule contains a sequence of amino acids set forth in SEQ ID NO: 361 or416 or a sequence of amino acids comprising at least 65% amino acidsequence identity to the sequence of amino acids set forth in SEQ ID NO:361 or 416 that specifically binds HA. For example, the link moduleexhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to the sequence of amino acids setforth in SEQ ID NO: 361 or 416 and specifically binds HA. In particularexamples, the link module contains a sequence of amino acids set forthin SEQ ID NO: 361 or 416.

Also provided herein is a TSG-6-LM multimer wherein the multimerizationdomain is selected from among an immunoglobulin constant region (Fc), aleucine zipper, complementary hydrophobic regions, complementaryhydrophilic regions, compatible protein-protein interaction domains,free thiols that form an intermolecular disulfide bond between twomolecules, and a protuberance-into-cavity and a compensatory cavity ofidentical or similar size that form stable multimers. In some examples,the multimerization domain is an Fc domain or a variant thereof thateffects multimerization. For example, the Fc domain is from an IgG, IgMor an IgE. In a particular example, the Fc domain has a sequence ofamino acids set forth in SEQ ID NO:359.

Also provided herein is a TSG-6-LM multimer containing a TSG-6 linkmodule and an immunoglobulin Fc domain. In some examples, the TSG-6-LMmultimer contains a sequence of amino acids set forth in SEQ ID NO: 212or 215 or a sequence of amino acids that exhibits at least 65% aminoacid sequence identity to SEQ ID NO:212 or 215. For example, the TSG-6multimer contains a sequence of amino acids that exhibits at least 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to the sequence of amino acids set forth in SEQ IDNO:212 or 215 and that specifically binds HA. In particular examples,the TSG-6-LM multimer contains a sequence of amino acids set forth inSEQ ID NO:212 or 215. In some examples, the TSG-6-LM multimer has abinding affinity to HA of at least 10⁷ M⁻¹, for example at least 1×10⁸M⁻¹, 2×10⁸ M⁻¹, 3×10⁸ M⁻¹, 4×10⁸ M⁻¹, 5×10⁸ M⁻¹, 6×10⁸ M⁻¹, 7×10⁸ M⁻¹,8×10⁸ M⁻¹, 9×10⁸ M⁻¹, 1×10⁹ M⁻¹ or lower.

Also provided herein are methods of selecting a subject, predictingefficacy and/or monitoring treatment using any of the above providedHABP to detect HA by in vivo imaging methods. The in vivo imaging methodcan be magnetic resonance imaging (MRI), single-photon emission computedtomography (SPECT), computed tomography (CT), computed axial tomography(CAT), electron beam computed tomography (EBCT), high resolutioncomputed tomography (HRCT), hypocycloidal tomography, positron emissiontomography (PET), scintigraphy, gamma camera, α β+ detector, a γdetector, fluorescence imaging, low-light imaging, X-rays, and/orbioluminescence imaging. In such methods, the HABP is conjugated,directly or indirectly, to a moiety that provides a signal or induces asignal that is detectable in vivo.

DETAILED DESCRIPTION Outline

-   -   A. DEFINITIONS    -   B. HYALURONAN BINDING PROTEIN AND COMPANION DIAGNOSTIC        -   1. Hyaluronan Accumulation in Disease And Correlation to            Prognosis        -   2. Therapy of Tumors with An Anti-Hyaluronan Agent (e.g.            Hyaluronan-Degrading Enzyme) and Responsiveness to Treatment        -   3. Hyaluronan Binding Proteins (HABPs) Reagent and            Diagnostic        -   4. Companion Diagnostic and Prognostic Methods    -   C. HYALURONAN BINDING PROTEINS (HABPs) FOR USE AS A COMPANION        DIAGNOSTIC        -   1. HA Binding Proteins with Link Modules or G1 domains            -   a. Type A: TSG-6 sub-group                -   i. TSG-6                -   ii. Stabilin-1 and Stabilin-2            -   b. Type B: CD44 sub-group                -   i. CD44                -   ii. LYVE-1            -   c. Type C: Link Protein sub-group                -   i. HAPLN/Link Protein family                -    (1) HAPLN1                -    (2) HAPLN2                -    (3) HAPLN3                -    (4) HAPLN4                -    (5) Aggrecan                -    (6) Brevican                -    (7) Versican                -    (8) Neurocan                -    (9) Phosphacan        -   2. HA Binding Proteins Without Link Modules            -   a. HABP1/C1QBP            -   b. Layilin            -   c. RHAMM            -   d. Others        -   3. Modifications of HA Binding Proteins            -   a. Multimers of HABP                -   i. Peptide Linkers                -   ii. Heterobifunctional linking agents                -   iii. Polypeptide Multimerization domains                -    (1) Immunoglobulin domain                -    (a) Fc domain                -    (2) Leucine Zipper                -    (3) Protein-Protein Interaction between Subunits                -   iv. Other multimerization domains            -   b. Mutations to Improve HA Binding            -   c. Modifications of HA Binding Proteins for Detection                -   i. Conjugation to Detectable Proteins or Moieties        -   4. Selection of HA Binding Proteins for Diagnostic Use    -   D. ASSAYS AND CLASSIFICATION        -   1. Assays for Measuring Hyaluronan            -   a. Histochemical and Immunohistochemical Methods            -   b. Solid Phase Binding Assays            -   c. In vivo Imaging Assays        -   2. Classification of Subjects    -   E. TREATMENT OF SELECTED SUBJECT WITH AN ANTI-HYALURONAN AGENT        -   1. Anti-Hyaluronan Agent            -   a. Agents that Inhibit Hyaluronan Synthesis            -   b. Hyaluronan-degrading Enzymes                -   i. Hyaluronidases                -    (1) Mammalian-type hyaluronidases                -    (a) PH20                -    (2) Other hyaluronidases                -    (3) Other hyaluronan degrading enzymes                -   ii. Soluble hyaluronan-degrading enzymes                -    (1) Soluble Human PH20                -    (2) rHuPH20                -   iii. Glycosylation of hyaluronan-degrading enzymes                -   iv. Modified (Polymer-Conjugated) hyaluronan                    degrading enzymes            -   2. Pharmaceutical Compositions and Formulations            -   3. Dosages and Administration                -   a. Administration of a PEGylated                    hyaluronan-degrading enzyme            -   4. Combination Treatments    -   F. METHODS OF PRODUCING NUCLEIC ACIDS AND ENCODED POLYPEPTIDES        OF HYALURONAN-DEGRADING ENZYMES AND HYALURONAN BINDING PROTEINS        -   1. Vectors and Cells        -   2. Expression            -   a. Prokaryotic Cells            -   b. Yeast Cells            -   c. Insect Cells            -   d. Mammalian Cells            -   e. Plants        -   3. Purification Techniques        -   4. PEGylation of Hyaluronan-degrading Enzyme Polypeptides    -   G. METHODS OF ASSESSING ACTIVITY AND MONITORING EFFECTS OF        ANTI-HYALURONAN AGENTS        -   1. Methods to Assess Side Effects        -   2. Evaluating Biomarkers Associated With Activity of an            Anti-Hyaluronan Agent (e.g. Hyaluronan-Degrading Enzyme            Activity)            -   a. Assays to assess the activity of a Hyaluronan                Degrading Enzyme            -   b. Measurement of HA catabolites            -   c. Tumor metabolic activity            -   d. Increased apparent diffusion and enhanced tumor                perfusion        -   3. Tumor Size and Volume        -   4. Pharmacokinetic and Pharmacodynamic Assays    -   H. KITS AND ARTICLES OF MANUFACTURE    -   I. EXAMPLES

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, GENBANK sequences, websites andother published materials referred to throughout the entire disclosureherein, unless noted otherwise, are incorporated by reference in theirentirety. In the event that there is a plurality of definitions forterms herein, those in this section prevail. Where reference is made toa URL or other such identifier or address, it is understood that suchidentifiers can change and particular information on the internet cancome and go, but equivalent information is known and can be readilyaccessed, such as by searching the internet and/or appropriatedatabases. Reference thereto evidences the availability and publicdissemination of such information.

As used herein, a companion diagnostic refers to a diagnostic method andor reagent that is used to identify subjects susceptible to treatmentwith a particular treatment or to monitor treatment and/or to identifyan effective dosage for a subject or sub-group or other group ofsubjects. For purposes herein, a companion diagnostic refers toreagents, such as modified TSG-6 proteins, that are used to detecthyaluronan in a sample. The companion diagnostic refers to the reagentsand also to the test(s) that is/are performed with the reagent.

As used herein, hyaluronan (HA; also known as hyaluronic acid orhyaluronate) refers to a naturally occurring polymer of repeateddisaccharide units of N-acetylglucosamine and D-glucuronic acid.Hyaluronan is produced by certain tumors.

As used herein, “high HA” with reference to the amount or level of HA ina tissue or body fluid sample refers to the degree or extent of HA inthe tissue or body fluid sample as compared to a normal or healthytissue or body fluid sample. The amount of HA is high if the amount isat least or at least about 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold or higher than the amount or level of HA in acorresponding normal or healthy tissue. It is understood that the amountof HA can be determined and quantitated or semi-quantitated usingmethods such as solid-phase binding assays or histochemistry. Forexample, the amount can be based on comparison of plasma levels orcomparison of staining intensity (e.g. percent positive pixels) asdetermined by histochemistry. For example, high HA exists if the HAscore by histochemistry or other method is HA⁺³ and/or if there is HAstaining over 25% of tumor section. For example, high HA exists if thereis a ratio of strong positive stain (such as brown stain) to the sum oftotal stained area that is more than 25% strong positive stain to totalstain the tumor tissue.

As used herein, an HA score refers to a semi-quantitative score of HApositivity levels on cell members and stroma of tumors. The score can bedetermined by detection of HA in tumor tissue, such as formalin-fixedand paraffin-embedded tissue, by histochemistry methods, such asimmunohistochemistry or pseudo immunohistochemistry methods, for HAusing an HABP. The degree of stain on cells and stroma can be determinedvisually under a microscope or by available computer algorithm programsand software. For example, images can be quantitatively analyzed using apixel count algorithm for HA stain (e.g. Aperio Spectrum Software andother standard methods that measure or quantitate or semi-quantitate thedegree of staining) A tumor is graded or scored as HA^(High) (HA⁺³) atstrong HA staining over 25% of tumor section; as HA^(Moderate) (HA⁺²) atstrong HA staining between 10 and 25% of tumor section; and as HA^(Low)(HA⁺¹) at strong HA staining under 10% of tumor section. For example, aratio of strong positive stain (such as brown stain) to the sum of totalstained area can be calculated and scored, where if the ratio is morethan 25% strong positive stain to total stain the tumor tissue is scoredas HA⁺³, if the ratio is 10-25% of strong positive stain to total stainthe tumor tissue is scored as HA⁺², if the ratio is less than 10% ofstrong positive stain to total stain the tumor tissue is scored as HA⁺¹,and if the ratio of strong positive stain to total stain is 0 the tumortissue is scored as 0. The Aperio method, as well as software therefor,are known to those of skill in the art (see, e.g., U.S. Pat. No.8,023,714; U.S. Pat. No. 7,257,268).

As used herein, a hyaluronan binding protein (HA binding protein; HABP)or hyaladherin refers to any protein that specifically binds to HA topermit detection of the HA. The binding affinity is one that has as anassociation constant Ka that is at least about or is at least 10⁷ M⁻¹.For the methods and companion diagnostic products provided herein, theHA binding protein is a recombinantly produced or synthetic protein(s),not a protein derived from a biological source or physiologic source,such as cartilage. HA binding proteins include HA binding domains,including link modules that bind to HA and sufficient portions thereofthat specifically binds to HA to permit detection thereof. Hence, HABPsinclude any protein that contains a hyaluronan binding region or domainor a sufficient portion thereof to specifically bind HA. Exemplaryhyaluronan binding regions are link modules (link domains) or G1domains. A sufficient portion includes at least 10, 20, 30, 40, 50, 60,70, 80, 90, 95 or more contiguous amino acids of a binding domain orlink module. HA binding proteins also include fusion proteins containingan HA binding protein and one or more additional polypeptides, includingmultimerization domains. Exemplary HA binding proteins include, but arenot limited to, aggrecan, versican, neurocan, brevican, phosphacan,TSG-6, TSG-6 mutants, such as those provided herein, includingpolypeptides containing HA binding domains and link modules thereof thatbind to HA.

As used herein, hyaluronan-binding domain or HA-binding domain refers toa region or domain of an HABP polypeptide that specifically binds tohyaluronan with a binding affinity that has as an association constantKa that is at least about or is at least 10⁶ M⁻¹ or 10⁷ M⁻¹ or greateror a dissociation constant Kd that is less than 10⁻⁶ M or 10⁻⁷ M orless. Exemplary hyaluronan-binding domains include, for example, linkmodules (also called link domains herein) or G1 domains, or sufficientportions of a link module or G1 domain that specifically binds to HA.

As used herein, reference that “the only portion of an HABP” is a linkmodule or G1 domain or grammatical variations thereof means that theHABP molecule (e.g. a TSG-6 link module) consists or consistsessentially of the link module or G1 domain but does not include thecomplete full-length sequence of amino acids of the reference HABP.Hence, the HABP only contains a hyaluronan-binding region or asufficient portion thereof to specifically bind to HA. It is understoodthat the HABP can contain additional non-HABP amino acid sequences,including but not limited to, sequences that correspond to a detectablemoiety or moiety capable of detection or a multimerization domain.

As used herein, modified, with respect to modified HA binding proteinsrefers to modifications to alter, typically improve, one more propertiesof an HA binding protein for detection in the diagnostic methodsprovided herein. Modifications include mutations that increase affinityand/or specificity of the protein for HA.

As used herein, a domain refers to a portion (a sequence of three ormore, generally 5 or 7 or more amino acids) of a polypeptide that is astructurally and/or functionally distinguishable or definable. Forexample, a domain includes those that can form an independently foldedstructure within a protein made up of one or more structural motifs(e.g. combinations of alpha helices and/or beta strands connected byloop regions) and/or that is recognized by virtue of a functionalactivity, such as kinase activity. A protein can have one, or more thanone, distinct domain. For example, a domain can be identified, definedor distinguished by homology of the sequence therein to related familymembers, such as homology and motifs that define an extracellulardomain. In another example, a domain can be distinguished by itsfunction, such as by enzymatic activity, e.g. kinase activity, or anability to interact with a biomolecule, such as DNA binding, ligandbinding, and dimerization. A domain independently can exhibit a functionor activity such that the domain independently or fused to anothermolecule can perform an activity, such as, for example proteolyticactivity or ligand binding. A domain can be a linear sequence of aminoacids or a non-linear sequence of amino acids from the polypeptide. Manypolypeptides contain a plurality of domains.

As used herein, a fusion protein refers to a chimeric protein containingtwo or more portions from two more proteins or peptides that are linkeddirectly or indirectly via peptide bonds.

As used herein, a multimerization domain refers to a sequence of aminoacids that promotes stable interaction of a polypeptide molecule withanother polypeptide molecule containing a complementary multimerizationdomain, which can be the same or a different multimerization domain toform a stable multimer with the first domains. Generally, a polypeptideis joined directly or indirectly to the multimerization domain.Exemplary multimerization domains include the immunoglobulin sequencesor portions thereof, leucine zippers, hydrophobic regions, hydrophilicregions, compatible protein-protein interaction domains such as, but notlimited to an R subunit of PKA and an anchoring domain (AD), a freethiol that forms an intermolecular disulfide bond between two molecules,and a protuberance-into-cavity (i.e., knob into hole) and a compensatorycavity of identical or similar size that form stable multimers. Themultimerization domain, for example, can be an immunoglobulin constantregion. The immunoglobulin sequence can be an immunoglobulin constantdomain, such as the Fc domain or portions thereof from IgG 1, IgG2, IgG3or IgG4 subtypes, IgA, IgE, IgD and IgM.

As used herein, “knobs into holes” (also referred to herein asprotuberance-into-cavity) refers to particular multimerization domainsengineered such that steric interactions between and/or among suchdomains, not only promote stable interaction, but also promote theformation of heterodimers (or multimers) over homodimers (orhomomultimers) from a mixture of monomers. This can be achieved, forexample by constructing protuberances and cavities. Protuberances can beconstructed by replacing small amino acid side chains from the interfaceof the first polypeptide with larger side chains (e.g. tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to theprotuberances optionally are created on the interface of a secondpolypeptide by replacing large amino acid side chains with smaller ones(e.g., alanine or threonine).

As used herein, complementary multimerization domains refer to two ormore multimerization domains that interact to form stable multimers ofpolypeptides linked to each such domain. Complementary multimerizationdomains can be the same domain or a member of a family of domains, suchas for example, Fc regions, leucine zippers, and knobs and holes.

As used herein, “Fc” or “Fc region” or “Fc domain” refers to apolypeptide containing the constant region of an antibody heavy chain,excluding the first constant region immunoglobulin domain. Thus, Fcrefers to the last two constant region immunoglobulin domains of IgA,IgD, and IgE, or the last three constant region immunoglobulin domainsof IgE and IgM. Optionally, an Fc domain can include all or part of theflexible hinge N-terminal to these domains. For IgA and IgM, Fc caninclude the J chain. For an exemplary Fc domain of IgG containsimmunoglobulin domains Cγ2 and Cγ3, and optionally all or part of thehinge between Cγ1 and Cγ2. The boundaries of the Fc region can vary, buttypically, include at least part of the hinge region. In addition, Fcalso includes any allelic or species variant or any variant or modifiedform, such as any variant or modified form that alters the binding to anFcR or alters an Fc-mediated effector function. Exemplary sequences ofother Fc domains, including modified Fc domains are known.

As used herein, “Fc chimera” refers to a chimeric polypeptide in whichone or more polypeptides is linked, directly or indirectly, to an Fcregion or a derivative thereof. Typically, an Fc chimera combines the Fcregion of an immunoglobulin with another polypeptide, such as forexample an ECD polypeptide. Derivatives of or modified Fc polypeptidesare known to those of skill in the art.

As used herein, “multimer” with reference to a hyaluronan bindingprotein refers to an HABP that contains multiple HA binding sites, forexample, at least 2, 3, or 4 HA binding sites. For example, an HABPmultimer refers to an HABP that contains at least 2 link modules thatare each capable of binding to HA. For example, a multimer can begenerated by linking, directly or indirectly, two or more link modules(e.g. TSG-6 link module). The linkage can be facilitated using amultimerization domain, such as an Fc protein.

As used herein, an allelic variant or allelic variation references to apolypeptide encoded by a gene that differs from a reference form of agene (i.e. is encoded by an allele). Typically the reference form of thegene encodes a wildtype form and/or predominant form of a polypeptidefrom a population or single reference member of a species. Typically,allelic variants, which include variants between and among speciestypically have at least 80%, 90% or greater amino acid identity with awildtype and/or predominant form from the same species; the degree ofidentity depends upon the gene and whether comparison is interspecies orintraspecies. Generally, intraspecies allelic variants have at leastabout 80%, 85%, 90% or 95% identity or greater with a wildtype and/orpredominant form, including 96%, 97%, 98%, 99% or greater identity witha wildtype and/or predominant form of a polypeptide.

As used herein, species variants refer to variants of the samepolypeptide between and among species. Generally, interspecies variantshave at least about 60%, 70%, 80%, 85%, 90%, or 95% identity or greaterwith a wildtype and/or predominant form from another species, including96%, 97%, 98%, 99% or greater identity with a wildtype and/orpredominant form of a polypeptide.

As used herein, modification in reference to modification of a sequenceof amino acids of a polypeptide or a sequence of nucleotides in anucleic acid molecule and includes deletions, insertions, andreplacements of amino acids and nucleotides, respectively.

As used herein, a composition refers to any mixture. It can be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, a combination refers to any association between or amongtwo or more items. The combination can be two or more separate items,such as two compositions or two collections, can be a mixture thereof,such as a single mixture of the two or more items, or any variationthereof. The elements of a combination are generally functionallyassociated or related. A kit is a packaged combination that optionallyincludes instructions for use of the combination or elements thereof.

As used herein, normal levels or values can be defined in a variety ofways known to one of skill in the art. Typically, normal levels refer tothe expression levels of an HA across a healthy population. The normallevels (or reference levels) are based on measurements of healthysubjects, such as from a specified source (i.e. blood, serum, tissue, orother source). Often, a normal level will be specified as a “normalrange”, which typically refers to the range of values of the median 95%of the healthy population. Reference value is used interchangeablyherein with normal level but can be different from normal levelsdepending on the subjects or the source. Reference levels are typicallydependent on the normal levels of a particular segment of thepopulation. Thus, for purposes herein, a normal or reference level is apredetermined standard or control by which a test patient can becompared.

As used herein, elevated level refers to the any level of amount orexpression of HA above a recited or normal threshold.

As used herein, biological sample refers to any sample obtained from aliving or viral source or other source of macromolecules andbiomolecules, and includes any cell type or tissue of a subject fromwhich nucleic acid or protein or other macromolecule can be obtained.The biological sample can be a sample obtained directly from abiological source or to sample that is processed. For example, isolatednucleic acids that are amplified constitute a biological sample.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples from animals, including biopsied tumorsamples.

As used herein, detection includes methods that permit visualization (byeye or equipment) of a protein. A protein can be visualized using anantibody specific to the protein. Detection of a protein can also befacilitated by fusion of a protein with a tag including an epitope tagor label.

As used herein, a label refers to a detectable compound or compositionwhich is conjugated directly or indirectly to a polypeptide so as togenerate a labeled polypeptide. The label can be detectable by itself(e.g., radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, can catalyze chemical alteration of a substratecompound composition which is detectable. Non-limiting examples oflabels included fluorogenic moieties, green fluorescent protein, orluciferase.

As used herein, affinity refers to the strength of interaction betweentwo molecules such as between a hyaluronan binding protein andhyaluronan. Affinity is often measured by equilibrium associationconstant (Ka) or equilibrium dissociation constant (Kd). The bindingaffinity between the molecules described herein, typically has a bindingaffinity with an association constant (Ka) of at least about 10⁶ l/mol,10⁷ l/mol, 10⁸ l/mol, 10⁹ l/mol or greater (generally 10⁷-10⁸ l/mol orgreater).

The binding affinity of molecules herein also can be described based onthe dissociation constant (Kd) of at least less than or less than or10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M or lower.

As used herein, reference to a sufficient portion thereof that binds toHA means that the binding molecule exhibits a Ka of at least or at leastabout 10⁷ to 10⁸ M⁻¹ or a dissociation constant (Kd) of 1×10⁻⁷ M or1×10⁻⁸ M or less to HA.

As used herein, specificity (also referred to herein as selectively)with respect to two molecules, such as with respect to a hyaluronanbinding protein and HA, refers to the greater affinity the two moleculesexhibit for each other compared to affinity for other molecules. Thus, ahyaluronan binding protein (HABP) with greater specificity for HA meansthat it binds to other molecules, such as heparin, with lower affinitythan it binds to HA. Specific binding typically results in selectivebinding.

As used herein, a “G1 domain” refers to an HA binding domain of a Type CHA binding protein. The G1 domain contains an Ig module and two linkmodules. Exemplary proteins that contain a G1 domain include HAPLN1/linkprotein, HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, brevican,neurocan and phosphacan.

As used herein, link modules or link domain, used interchangeablyherein, are hyaluronan-binding domains that occur in proteins andfacilitate binding to HA and that are involved in the assembly ofextracellular matrix, cell adhesion and migration. For example, the linkmodule from human TSG-6 contains two alpha helices and two antiparallelbeta sheets arranged around a hydrophobic core. This defines theconsensus fold for the Link module superfamily, which includes CD44,TSG-6, cartilage link protein, aggrecan and others as described herein.

As used herein, an “Ig module” refers to the portion of the G1 domain ofType C HABPs that is involved in the binding between Type C HABPs. Igmodules of Type C hyaluronans interact with one another to form a stabletertiary structure with hyaluronan.

As used herein, a “solid phase binding assay” refers to an in vitroassay in which an antigen is contacted with a ligand, where one of theantigen or ligand are bound to a solid support. The solid phase can beone in which components are physically immobilized to a solid support.For example, solid supports include, but are not limited to, amicrotiter plate, a membrane (e.g., nitrocellulose), a bead, a dipstick,a thin-layer chromatographic plate, or other solid medium. Uponantigen-ligand interaction, the unwanted or non-specific components canbe removed (e.g. by washing) and the antigen-ligand complex detected.

As used herein, predicting efficacy of treatment with an anti-hyaluronanagent, such as a hyaluronan-degrading enzyme, means that the companiondiagnostic can be a prognostic indicator of treatment with ananti-hyaluronan agent, such as a hyaluronan degrading enzyme. Forexample, based on the results of detection of hyaluronan or other markerwith the companion diagnostic, it can be determined that ananti-hyaluronan agent, such as a hyaluronan-degrading enzyme, willlikely have some effect in treating subject.

As used herein, a prognostic indicator refers to a parameter thatindicates the probability of a particular outcome, such as theprobability that a treatment will be effective for a particular diseaseor subject.

As used herein, elevated HA in a sample refers to an amount of HA in asample that is increased compared to the level present in acorresponding sample from a healthy sample or compared to apredetermined standard.

As used herein, elevated hyaluronan levels refers to amounts ofhyaluronan in particular tissue, body fluid or cell, dependent upon thedisease or condition, as a consequence of or otherwise observed in thedisease. For example, as consequence of the presence of ahyaluronan-rich tumor, hyaluronan (HA) levels can be elevated in bodyfluids, such as blood, urine, saliva and serum, and/or in the tumoroustissue or cell. The level can be compared to a standard or othersuitable control, such as a comparable sample from a subject who doesnot have the HA-associated disease, such as a subject that does not havea tumor.

As used herein, corresponding residues refers to residues that occur ataligned loci. Related or variant polypeptides are aligned by any methodknown to those of skill in the art. Such methods typically maximizematches, and include methods such as using manual alignments and byusing the numerous alignment programs available (for example, BLASTP)and others known to those of skill in the art. By aligning the sequencesof polypeptides, one skilled in the art can identify correspondingresidues, using conserved and identical amino acid residues as guides.Corresponding positions also can be based on structural alignments, forexample by using computer simulated alignments of protein structure. Inother instances, corresponding regions can be identified.

As used herein, an anti-hyaluronan agent refers to any agent thatmodulates hyaluronan (HA) synthesis or degradation, thereby alteringhyaluronan levels in a tissue or cell. For purposes herein,anti-hyaluronan agents reduce hyaluronan levels in a tissue or cellcompared to the absence of the agent. Such agents include compounds thatmodulate the expression of genetic material encoding HA synthase (HAS)and other enzymes or receptors involved in hyaluronan metabolism, orthat modulate the proteins that synthesize or degrade hyaluronanincluding HAS function or activity. The agents include small-molecules,nucleic acids, peptides, proteins or other compounds. For example,anti-hyaluronan agents include, but are not limited to, antisense orsense molecules, antibodies, enzymes, small molecule inhibitors and HASsubstrate analogs.

As used herein, a hyaluronan-degrading enzyme refers to an enzyme thatcatalyzes the cleavage of a hyaluronan polymer (also referred to ashyaluronic acid or HA) into smaller molecular weight fragments.Exemplary of hyaluronan-degrading enzymes are hyaluronidases, andparticular chondroitinases and lyases that have the ability todepolymerize hyaluronan. Exemplary chondroitinases that arehyaluronan-degrading enzymes include, but are not limited to,chondroitin ABC lyase (also known as chondroitinase ABC), chondroitin AClyase (also known as chondroitin sulfate lyase or chondroitin sulfateeliminase) and chondroitin C lyase. Chondroitin ABC lyase comprises twoenzymes, chondroitin-sulfate-ABC endolyase (EC 4.2.2.20) andchondroitin-sulfate-ABC exolyase (EC 4.2.2.21). An exemplarychondroitin-sulfate-ABC endolyases and chondroitin-sulfate-ABC exolyasesinclude, but are not limited to, those from Proteus vulgaris andPedobacter heparinus (the Proteus vulgaris chondroitin-sulfate-ABCendolyase is set forth in SEQ ID NO:98; Sato et al. (1994) Appl.Microbiol. Biotechnol. 41(1):39-46). Exemplary chondroitinase AC enzymesfrom the bacteria include, but are not limited to, those from Pedobacterheparinus, set for th in SEQ ID NO: 99, Victivallis vadensis, set forthin SEQ ID NO:100, and Arthrobacter aurescens (Tkalec et al. (2000)Applied and Environmental Microbiology 66(1):29-35; Ernst et al. (1995)Critical Reviews in Biochemistry and Molecular Biology 30(5):387-444).Exemplary chondroitinase C enzymes from the bacteria include, but arenot limited to, those from Streptococcus and Flavobacterium (Hibi et al.(1989) FEMS-Microbiol-Lett. 48(2):121-4; Michelacci et al. (1976) J.Biol. Chem. 251:1154-8; Tsuda et al. (1999) Eur. J. Biochem.262:127-133).

As used herein, hyaluronidase refers to a class of hyaluronan-degradingenzymes. Hyaluronidases include bacterial hyaluronidases (EC 4.2.2.1 orEC 4.2.99.1), hyaluronidases from leeches, other parasites, andcrustaceans (EC 3.2.1.36), and mammalian-type hyaluronidases (EC3.2.1.35). Hyaluronidases include any of non-human origin including, butnot limited to, murine, canine, feline, leporine, avian, bovine, ovine,porcine, equine, piscine, ranine, bacterial, and any from leeches, otherparasites, and crustaceans. Exemplary non-human hyaluronidases include,hyaluronidases from cows (SEQ ID NOS:10, 11, 64 and BH55 (U.S. Pat. Nos.5,747,027 and 5,827,721), yellow jacket wasp (SEQ ID NOS:12 and 13),honey bee (SEQ ID NO:14), white-face hornet (SEQ ID NO:15), paper wasp(SEQ ID NO:16), mouse (SEQ ID NOS:17-19, 32), pig (SEQ ID NOS:20-21),rat (SEQ ID NOS:22-24, 31), rabbit (SEQ ID NO:25), sheep (SEQ ID NOS:26,27, 63 and 65), chimpanzee (SEQ ID NO:101), Rhesus monkey (SEQ IDNO:102), orangutan (SEQ ID NO:28), cynomolgus monkey (SEQ ID NO:29),guinea pig (SEQ ID NO:30), Arthrobacter sp. (strain FB24 (SEQ IDNO:67)), Bdellovibrio bacteriovorus (SEQ ID NO:68), Propionibacteriumacnes (SEQ ID NO:69), Streptococcus agalactiae ((SEQ ID NO:70); 18RS21(SEQ ID NO:71); serotype Ia (SEQ ID NO:72); serotype III (SEQ IDNO:73)), Staphylococcus aureus (strain COL (SEQ ID NO:74); strainMRSA252 (SEQ ID NOS:75 and 76); strain MSSA476 (SEQ ID NO:77); strainNCTC 8325 (SEQ ID NO:78); strain bovine RF122 (SEQ ID NOS:79 and 80);strain USA300 (SEQ ID NO:81)), Streptococcus pneumoniae ((SEQ ID NO:82);strain ATCC BAA-255/R6 (SEQ ID NO:83); serotype 2, strain D39/NCTC 7466(SEQ ID NO:84)), Streptococcus pyogenes (serotype M1 (SEQ ID NO:85);serotype M2, strain MGAS 10270 (SEQ ID NO:86); serotype M4, strain MGAS10750 (SEQ ID NO:87); serotype M6 (SEQ ID NO:88); serotype M12, strainMGAS2096 (SEQ ID NOS:89 and 90); serotype M12, strain MGAS9429 (SEQ IDNO:91); serotype M28 (SEQ ID NO:92)), Streptococcus suis (SEQ IDNOS:93-95); Vibrio fischeri (strain ATCC 700601/ES114 (SEQ ID NO:96)),and the Streptomyces hyaluronolyticus hyaluronidase enzyme, which isspecific for hyaluronic acid and does not cleave chondroitin orchondroitin sulfate (Ohya, T. and Kaneko, Y. (1970) Biochim. Biophys.Acta 198:607). Hyaluronidases also include those of human origin.Exemplary human hyaluronidases include HYAL1 (SEQ ID NO:36), HYAL2 (SEQID NO:37), HYAL3 (SEQ ID NO:38), HYAL4 (SEQ ID NO:39), and PH20 (SEQ IDNO:1). Also included amongst hyaluronidases are soluble hyaluronidases,including, ovine and bovine PH20, soluble human PH20 and solublerHuPH20. Examples of commercially available bovine or ovine solublehyaluronidases include Vitrase® (ovine hyaluronidase), Amphadase®(bovine hyaluronidase) and Hydase™ (bovine hyaluronidase).

As used herein, “purified bovine testicular hyaluronidase” refers to abovine hyaluronidase purified from bovine testicular extracts (see U.S.Pat. Nos. 2,488,564, 2,488,565, 2,806,815, 2,808,362, 2,676,139,2,795,529, 5,747,027 and 5,827,721). Examples of commercially availablepurified bovine testicular hyaluronidases include Amphadase® andHydase™, and bovine hyaluronidases, including, but not limited to, thoseavailable from Sigma Aldrich, Abnova, EMD Chemicals, GenWay Biotech,Inc., Raybiotech, Inc., and Calzyme. Also included are recombinantlyproduced bovine hyaluronidases, such as but not limited to, thosegenerated by expression of a nucleic acid molecule set forth in any ofSEQ ID NOS:190-192.

As used herein, “purified ovine testicular hyaluronidase” refers to anovine hyaluronidase purified from ovine testicular extracts (see U.S.Pat. Nos. 2,488,564, 2,488,565 and 2,806,815 and International PCTApplication No. WO2005/118799). Examples of commercially availablepurified ovine testicular extract include Vitrase®, and ovinehyaluronidases, including, but not limited to, those available fromSigma Aldrich, Cell Sciences, EMD Chemicals, GenWay Biotech, Inc.,Mybiosource.com and Raybiotech, Inc. Also included are recombinantlyproduced ovine hyaluronidases, such as, but not limited to, thosegenerated by expression of a nucleic acid molecule set forth in any ofSEQ ID NOS:66 and 193-194.

As used herein, “PH20” refers to a type of hyaluronidase that occurs insperm and is neutral-active. PH-20 occurs on the sperm surface, and inthe lysosome-derived acrosome, where it is bound to the inner acrosomalmembrane. PH20 includes those of any origin including, but not limitedto, human, chimpanzee, Cynomolgus monkey, Rhesus monkey, murine, bovine,ovine, guinea pig, rabbit and rat origin. Exemplary PH20 polypeptidesinclude those from human (SEQ ID NO:1), chimpanzee (SEQ ID NO:101),Rhesus monkey (SEQ ID NO:102), Cynomolgus monkey (SEQ ID NO:29), cow(SEQ ID NOS:11 and 64), mouse (SEQ ID NO:32), rat (SEQ ID NO:31), rabbit(SEQ ID NO:25), sheep (SEQ ID NOS:27, 63 and 65) and guinea pig (SEQ IDNO:30).

Reference to hyaluronan-degrading enzymes includes precursorhyaluronan-degrading enzyme polypeptides and mature hyaluronan-degradingenzyme polypeptides (such as those in which a signal sequence has beenremoved), truncated forms thereof that have activity, and includesallelic variants and species variants, variants encoded by splicevariants, and other variants, including polypeptides that have at least40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more sequence identity to the precursor polypeptides set forth inSEQ ID NOS: 1 and 10-48, 63-65, 67-102, or the mature forms thereof. Forexample, reference to hyaluronan-degrading enzyme also includes thehuman PH20 precursor polypeptide variants set forth in SEQ ID NOS:50-51.Hyaluronan-degrading enzymes also include those that contain chemical orposttranslational modifications and those that do not contain chemicalor posttranslational modifications. Such modifications include, but arenot limited to, PEGylation, albumination, glycosylation, farnesylation,carboxylation, hydroxylation, phosphorylation, and other polypeptidemodifications known in the art. A truncated PH20 hyaluronidase is anyC-terminal shortened form thereof, particularly forms that are truncatedand neutral active when N-glycosylated.

As used herein, a “soluble PH20” refers to any form of PH20 that issoluble under physiologic conditions. A soluble PH20 can be identified,for example, by its partitioning into the aqueous phase of a Triton®X-114 solution at 37° C. (Bordier et al., (1981) J. Biol. Chem.,256:1604-7). Membrane-anchored PH20, such as lipid-anchored PH20,including GPI-anchored PH20, will partition into the detergent-richphase, but will partition into the detergent-poor or aqueous phasefollowing treatment with Phospholipase-C. Included among soluble PH20are membrane-anchored PH20 in which one or more regions associated withanchoring of the PH20 to the membrane has been removed or modified,where the soluble form retains hyaluronidase activity. Soluble PH20 alsoinclude recombinant soluble PH20 and those contained in or purified fromnatural sources, such as, for example, testes extracts from sheep orcows. Exemplary of such soluble PH20 is soluble human PH20.

As used herein, soluble human PH20 or sHuPH20 includes PH20 polypeptideslacking all or a portion of the glycosylphosphatidylinositol (GPI)anchor sequence at the C-terminus such that upon expression, thepolypeptides are soluble under physiological conditions. Solubility canbe assessed by any suitable method that demonstrates solubility underphysiologic conditions. Exemplary of such methods is the Triton® X-114assay, that assesses partitioning into the aqueous phase and that isdescribed above and in the examples. In addition, a soluble human PH20polypeptide is, if produced in CHO cells, such as CHO-S cells, apolypeptide that is expressed and is secreted into the cell culturemedium. Soluble human PH20 polypeptides, however, are not limited tothose produced in CHO cells, but can be produced in any cell or by anymethod, including recombinant expression and polypeptide synthesis.Reference to secretion in CHO cells is definitional. Hence, if apolypeptide could be expressed and secreted in CHO cells and is soluble,i.e. partitions into the aqueous phase when extracted with Triton®X-114, it is a soluble PH20 polypeptide whether or not it isso-produced. The precursor polypeptides for sHuPH20 polypeptides caninclude a signal sequence, such as a heterologous or non-heterologous(i.e. native) signal sequence. Exemplary of the precursors are thosethat include a signal sequence, such as the native 35 amino acid signalsequence at amino acid positions 1-35 (see, e.g., amino acids 1-35 ofSEQ ID NO:1).

As used herein, an “extended soluble PH20” or “esPH20” includes solublePH20 polypeptides that contain residues up to the GPI anchor-attachmentsignal sequence and one or more contiguous residues from the GPI-anchorattachment signal sequence such that the esPH20 is soluble underphysiological conditions. Solubility under physiological conditions canbe determined by any method known to those of skill in the art. Forexample, it can be assessed by the Triton® X-114 assay described aboveand in the examples. In addition, as discussed above, a soluble PH20 is,if produced in CHO cells, such as CHO-S cells, a polypeptide that isexpressed and is secreted into the cell culture medium. Soluble humanPH20 polypeptides, however, are not limited to those produced in CHOcells, but can be produced in any cell or by any method, includingrecombinant expression and polypeptide synthesis. Reference to secretionin CHO cells is definitional. Hence, if a polypeptide could be expressedand secreted in CHO cells and is soluble, i.e. partitions into theaqueous phase when extracted with Triton® X-114, it is a soluble PH20polypeptide whether or not it is so-produced. Human soluble esPH20polypeptides include, in addition to residues 36-490, one or morecontiguous amino acids from amino acid residue position 491 of SEQ IDNO:1, inclusive, such that the resulting polypeptide is soluble.Exemplary human esPH20 soluble polypeptides are those that have aminoacids residues corresponding to amino acids 36-491, 36-492, 36-493,36-494, 36-495, 36-496 and 36-497 of SEQ ID NO:1. Exemplary of these arethose with an amino acid sequence set forth in any of SEQ ID NOS:151-154and 185-187. Also included are allelic variants and other variants, suchas any with 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with thecorresponding polypeptides of SEQ ID NOS:151-154 and 185-187 that retainneutral activity and are soluble. Reference to sequence identity refersto variants with amino acid substitutions.

As used herein, reference to “esPH20s” includes precursor esPH20polypeptides and mature esPH20 polypeptides (such as those in which asignal sequence has been removed), truncated forms thereof that haveenzymatic activity (retaining at least 1%, 10%, 20%, 30%, 40%, 50% ormore of the full-length form) and are soluble, and includes allelicvariants and species variants, variants encoded by splice variants, andother variants, including polypeptides that have at least 40%, 45%, 50%,55%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to the precursor polypeptides setforth in SEQ ID NOS:1 and 3, or the mature forms thereof.

As used herein, reference to “esPH20s” also include those that containchemical or posttranslational modifications and those that do notcontain chemical or posttranslational modifications. Such modificationsinclude, but are not limited to, PEGylation, albumination,glycosylation, farnesylation, carboxylation, hydroxylation,phosphorylation, and other polypeptide modifications known in the art.

As used herein, “soluble recombinant human PH20 (rHuPH20)” refers to acomposition containing solubles form of human PH20 as recombinantlyexpressed and secreted in Chinese Hamster Ovary (CHO) cells. SolublerHuPH20 is encoded by nucleic acid molecule that includes a signalsequence and is set forth in SEQ ID NO:49. The nucleic acid encodingsoluble rHuPH20 is expressed in CHO cells which secrete the maturepolypeptide. As produced in the culture medium, there is heterogeneityat the C-terminus so that the product includes a mixture of species thatcan include any one or more amino acids 36-481 and 36-482 of PH20 (e.g.,SEQ ID NO:4 to SEQ ID NO:9) in various abundance.

Similarly, for other forms of PH20, such as the esPH20s, recombinantlyexpressed polypeptides and compositions thereof can include a pluralityof species whose C-terminus exhibits heterogeneity. For example,compositions of recombinantly expressed esPH20 produced by expression ofthe polypeptide of SEQ ID NO:151, which encodes an esPH20 that has aminoacids 36-497, can include forms with fewer amino acids, such as 36-496,36-495.

As used herein, an “N-linked moiety” refers to an asparagine (N) aminoacid residue of a polypeptide that is capable of being glycosylated bypost-translational modification of a polypeptide. Exemplary N-linkedmoieties of human PH20 include amino acids N82, N166, N235, N254, N368and N393 of human PH20 set forth in SEQ ID NO:1.

As used herein, an “N-glycosylated polypeptide” refers to a PH20polypeptide or truncated form thereto containing oligosaccharide linkageof at least three N-linked amino acid residues, for example, N-linkedmoieties corresponding to amino acid residues N235, N368 and N393 of SEQID NO: 1. An N-glycosylated polypeptide can include a polypeptide wherethree, four, five and up to all of the N-linked moieties are linked toan oligosaccharide. The N-linked oligosaccharides can includeoligomannose, complex, hybrid or sulfated oligosaccharides, or otheroligosaccharides and monosaccharides.

As used herein, an “N-partially glycosylated polypeptide” refers to apolypeptide that minimally contains an N-acetylglucosamine glycan linkedto at least three N-linked moieties. A partially glycosylatedpolypeptide can include various glycan forms, including monosaccharides,oligosaccharides, and branched sugar forms, including those formed bytreatment of a polypeptide with EndoH, EndoF1, EndoF2 and/or EndoF3.

As used herein, a “deglycosylated PH20 polypeptide” refers to a PH20polypeptide in which fewer than all possible glycosylation sites areglycosylated. Deglycosylation can be effected, for example, by removingglycosylation, by preventing it, or by modifying the polypeptide toeliminate a glycosylation site. Particular N-glycosylation sites are notrequired for activity, whereas others are.

As used herein, “PEGylated” refers to covalent or other stableattachment of polymeric molecules, such as polyethylene glycol(PEGylation moiety PEG) to hyaluronan-degrading enzymes, such ashyaluronidases, typically to increase half-life of thehyaluronan-degrading enzyme.

As used herein, a “conjugate” refers to a polypeptide linked directly orindirectly to one or more other polypeptides or chemical moieties. Suchconjugates include fusion proteins, those produced by chemicalconjugates and those produced by any other methods. For example, aconjugate refers to soluble PH20 polypeptides linked directly orindirectly to one or more other polypeptides or chemical moieties,whereby at least one soluble PH20 polypeptide is linked, directly orindirectly to another polypeptide or chemical moiety so long as theconjugate retains hyaluronidase activity.

As used herein, a “fusion” protein refers to a polypeptide encoded by anucleic acid sequence containing a coding sequence from one nucleic acidmolecule and the coding sequence from another nucleic acid molecule inwhich the coding sequences are in the same reading frame such that whenthe fusion construct is transcribed and translated in a host cell, theprotein is produced containing the two proteins. The two molecules canbe adjacent in the construct or separated by a linker polypeptide thatcontains, 1, 2, 3, or more, but typically fewer than 10, 9, 8, 7, or 6amino acids. The protein product encoded by a fusion construct isreferred to as a fusion polypeptide.

As used herein, a “polymer” refers to any high molecular weight naturalor synthetic moiety that is conjugated to, i.e. stably linked directlyor indirectly via a linker, to a polypeptide. Such polymers, typicallyincrease serum half-life, and include, but are not limited to sialicmoieties, PEGylation moieties, dextran, and sugar and other moieties,such as for glycosylation. For example, hyaluronidases, such as asoluble PH20 or rHuPH20, can be conjugated to a polymer.

As used herein, a hyaluronidase substrate refers to a substrate (e.g.protein or polysaccharide) that is cleaved and/or depolymerized by ahyaluronidase enzyme. Generally, a hyaluronidase substrate is aglycosaminoglycan. An exemplary hyaluronidase substrate is hyaluronan(HA).

As used herein, a hyaluronan-associated disease, disorder or conditionrefers to any disease or condition in which hyaluronan levels areelevated as cause, consequence or otherwise observed in the disease orcondition. Hyaluronan-associated diseases and conditions are associatedwith elevated hyaluronan expression in a tissue or cell, increasedinterstitial fluid pressure, decreased vascular volume, and/or increasedwater content in a tissue. Hyaluronan-associated diseases, disorders orconditions can be treated by administration of a composition containingan anti-hyaluronan agent, such as a hyaluronan-degrading enzyme, such asa hyaluronidase, for example, a soluble hyaluronidase, either alone orin combination with or in addition to another treatment and/or agent.Exemplary diseases and conditions, include, but are not limited to,inflammatory diseases and hyaluronan-rich cancers. Hyaluronan richcancers include, for example, tumors, including solid tumors such aslate-stage cancers, a metastatic cancers, undifferentiated cancers,ovarian cancer, in situ carcinoma (ISC), squamous cell carcinoma (SCC),prostate cancer, pancreatic cancer, non-small cell lung cancer, breastcancer, colon cancer and other cancers.

Also exemplary of hyaluronan-associated diseases and conditions arediseases that are associated with elevated interstitial fluid pressure,such as diseases associated with disc pressure, and edema, for example,edema caused by organ transplant, stroke, brain trauma or other injury.Exemplary hyaluronan-associated diseases and conditions include diseasesand conditions associated with elevated interstitial fluid pressure,decreased vascular volume, and/or increased water content in a tissue,including cancers, disc pressure and edema. In one example, treatment ofthe hyaluronan-associated condition, disease or disorder includesamelioration, reduction, or other beneficial effect on one or more ofincreased interstitial fluid pressure (IFP), decreased vascular volume,and increased water content in a tissue.

As used herein, “activity” refers to a functional activity or activitiesof a polypeptide or portion thereof associated with a full-length(complete) protein. For example, active fragments of a polypeptide canexhibit an activity of a full-length protein. Functional activitiesinclude, but are not limited to, biological activity, catalytic orenzymatic activity, antigenicity (ability to bind or compete with apolypeptide for binding to an anti-polypeptide antibody),immunogenicity, ability to form multimers, and the ability tospecifically bind to a receptor or ligand for the polypeptide.

As used herein, “hyaluronidase activity” refers to the ability toenzymatically catalyze the cleavage of hyaluronic acid. The UnitedStates Pharmacopeia (USP) XXII assay for hyaluronidase determineshyaluronidase activity indirectly by measuring the amount of highermolecular weight hyaluronic acid, or hyaluronan, (HA) substrateremaining after the enzyme is allowed to react with the HA for 30 min at37° C. (USP XXII-NF XVII (1990) 644-645 United States PharmacopeiaConvention, Inc, Rockville, Md.). A Reference Standard solution can beused in an assay to ascertain the relative activity, in units, of anyhyaluronidase. In vitro assays to determine the hyaluronidase activityof hyaluronidases, such as PH20, including soluble PH20 and esPH20, areknown in the art and described herein. Exemplary assays include themicroturbidity assay that measures cleavage of hyaluronic acid byhyaluronidase indirectly by detecting the insoluble precipitate formedwhen the uncleaved hyaluronic acid binds with serum albumin and thebiotinylated-hyaluronic acid assay that measures the cleavage ofhyaluronic acid indirectly by detecting the remainingbiotinylated-hyaluronic acid non-covalently bound to microtiter platewells with a streptavidin-horseradish peroxidase conjugate and achromogenic substrate. Reference Standards can be used, for example, togenerate a standard curve to determine the activity in Units of thehyaluronidase being tested.

As used herein, specific activity refers to Units of activity per mgprotein. The milligrams of hyaluronidase is defined by the absorption ofa solution of at 280 nm assuming a molar extinction coefficient ofapproximately 1.7, in units of M⁻¹ cm⁻¹.

As used herein, “neutral active” refers to the ability of a PH20polypeptide to enzymatically catalyze the cleavage of hyaluronic acid atneutral pH (e.g. at or about pH 7.0). Generally, a neutral active andsoluble PH20, e.g., C-terminally truncated or N-partially glycosylatedPH20, has or has about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 200%,300%, 400%, 500%, 1000% or more activity compared to the hyaluronidaseactivity of a corresponding neutral active PH20 that is not C-terminallytruncated or N-partially glycosylated.

As used herein, a “GPI-anchor attachment signal sequence” is aC-terminal sequence of amino acids that directs addition of a preformedGPI-anchor to the polypeptide within the lumen of the ER. GPI-anchorattachment signal sequences are present in the precursor polypeptides ofGPI-anchored polypeptides, such as GPI-anchored PH20 polypeptides. TheC-terminal GPI-anchor attachment signal sequence typically contains apredominantly hydrophobic region of 8-20 amino acids, preceded by ahydrophilic spacer region of 8-12 amino acids, immediately downstream ofthe w-site, or site of GPI-anchor attachment. GPI-anchor attachmentsignal sequences can be identified using methods well known in the art.These include, but are not limited to, in silico methods and algorithms(see, e.g. Udenfriend et al. (1995) Methods Enzymol. 250:571-582,Eisenhaber et al., (1999) J. Biol. Chem. 292: 741-758, Fankhauser etal., (2005) Bioinformatics 21:1846-1852, Omaetxebarria et al., (2007)Proteomics 7:1951-1960, Pierleoni et al., (2008) BMC Bioinformatics9:392), including those that are readily available on bioinformaticwebsites, such as the ExPASy Proteomics tools site (e.g., theWorldWideWeb site expasy.ch/tools/).

As used herein, “nucleic acids” include DNA, RNA and analogs thereof,including peptide nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double-stranded. When referring to probes orprimers, which are optionally labeled, such as with a detectable label,such as a fluorescent or radiolabel, single-stranded molecules arecontemplated. Such molecules are typically of a length such that theirtarget is statistically unique or of low copy number (typically lessthan 5, generally less than 3) for probing or priming a library.Generally a probe or primer contains at least 14, 16 or 30 contiguousnucleotides of sequence complementary to or identical to a gene ofinterest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleicacids long.

As used herein, a peptide refers to a polypeptide that is greater thanor equal to 2 amino acids in length, and less than or equal to 40 aminoacids in length.

As used herein, the amino acids which occur in the various sequences ofamino acids provided herein are identified according to their known,three-letter or one-letter abbreviations (Table 1). The nucleotideswhich occur in the various nucleic acid fragments are designated withthe standard single-letter designations used routinely in the art.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids include the twentynaturally-occurring amino acids, non-natural amino acids and amino acidanalogs (i.e., amino acids wherein the α-carbon has a side chain).

As used herein, “amino acid residue” refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are presumed to be inthe “L” isomeric form. Residues in the “D” isomeric form, which are sodesignated, can be substituted for any L-amino acid residue as long asthe desired functional property is retained by the polypeptide. NH2refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxyl terminus of a polypeptide. In keeping with standard polypeptidenomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), andadopted 37 C.F.R. §§1.821-1.822, abbreviations for amino acid residuesare shown in Table 1:

TABLE 1 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu Glutamic acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine DAsp Aspartic acid N Asn Asparagine B Asx Asn and/or Asp C Cys Cysteine XXaa Unknown or other

All amino acid residue sequences represented herein by formulae have aleft to right orientation in the conventional direction ofamino-terminus to carboxyl-terminus. In addition, the phrase “amino acidresidue” is defined to include the amino acids listed in the Table ofCorrespondence (Table 1) and modified and unusual amino acids, such asthose referred to in 37 C.F.R. §§1.821-1.822, and incorporated herein byreference. Furthermore, it should be noted that a dash at the beginningor end of an amino acid residue sequence indicates a peptide bond to afurther sequence of one or more amino acid residues, to anamino-terminal group such as NH₂ or to a carboxyl-terminal group such asCOOH.

As used herein, the “naturally occurring α-amino acids” are the residuesof those 20 α-amino acids found in nature which are incorporated intoprotein by the specific recognition of the charged tRNA molecule withits cognate mRNA codon in humans. Non-naturally occurring amino acidsthus include, for example, amino acids or analogs of amino acids otherthan the 20 naturally-occurring amino acids and include, but are notlimited to, the D-stereoisomers of amino acids. Exemplary non-naturalamino acids are described herein and are known to those of skill in theart.

As used herein, a DNA construct is a single- or double-stranded, linearor circular DNA molecule that contains segments of DNA combined andjuxtaposed in a manner not found in nature. DNA constructs exist as aresult of human manipulation, and include clones and other copies ofmanipulated molecules.

As used herein, a DNA segment is a portion of a larger DNA moleculehaving specified attributes. For example, a DNA segment encoding aspecified polypeptide is a portion of a longer DNA molecule, such as aplasmid or plasmid fragment, which, when read from the 5′ to 3′direction, encodes the sequence of amino acids of the specifiedpolypeptide.

As used herein, the term polynucleotide means a single- ordouble-stranded polymer of deoxyribonucleotides or ribonucleotide basesread from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, andcan be isolated from natural sources, synthesized in vitro, or preparedfrom a combination of natural and synthetic molecules. The length of apolynucleotide molecule is given herein in terms of nucleotides(abbreviated “nt”) or base pairs (abbreviated “bp”). The termnucleotides is used for single- and double-stranded molecules where thecontext permits. When the term is applied to double-stranded moleculesit is used to denote overall length and will be understood to beequivalent to the term base pairs. It will be recognized by thoseskilled in the art that the two strands of a double-strandedpolynucleotide can differ slightly in length and that the ends thereofcan be staggered; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will, ingeneral, not exceed 20 nucleotides in length.

As used herein, “similarity” between two proteins or nucleic acidsrefers to the relatedness between the sequence of amino acids of theproteins or the nucleotide sequences of the nucleic acids. Similaritycan be based on the degree of identity and/or homology of sequences ofresidues and the residues contained therein. Methods for assessing thedegree of similarity between proteins or nucleic acids are known tothose of skill in the art. For example, in one method of assessingsequence similarity, two amino acid or nucleotide sequences are alignedin a manner that yields a maximal level of identity between thesequences. “Identity” refers to the extent to which the amino acid ornucleotide sequences are invariant. Alignment of amino acid sequences,and to some extent nucleotide sequences, also can take into accountconservative differences and/or frequent substitutions in amino acids(or nucleotides). Conservative differences are those that preserve thephysico-chemical properties of the residues involved. Alignments can beglobal (alignment of the compared sequences over the entire length ofthe sequences and including all residues) or local (the alignment of aportion of the sequences that includes only the most similar region orregions).

“Identity” per se has an art-recognized meaning and can be calculatedusing published techniques. (See, e.g. Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991). While there exists a number of methodsto measure identity between two polynucleotide or polypeptides, the term“identity” is well known to skilled artisans (Carrillo, H. and Lipton,D., (1988) SIAM J Applied Math 48:1073).

As used herein, homologous (with respect to nucleic acid and/or aminoacid sequences) means about greater than or equal to 25% sequencehomology, typically greater than or equal to 25%, 40%, 50%, 60%, 70%,80%, 85%, 90% or 95% sequence homology; the precise percentage can bespecified if necessary. For purposes herein the terms “homology” and“identity” are often used interchangeably, unless otherwise indicated.In general, for determination of the percentage homology or identity,sequences are aligned so that the highest order match is obtained (see,e.g.: Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; Carrillo, H. and Lipton, D., (1988) SIAM J Applied Math 48:1073).By sequence homology, the number of conserved amino acids is determinedby standard alignment algorithms programs, and can be used with defaultgap penalties established by each supplier. Substantially homologousnucleic acid molecules would hybridize typically at moderate stringencyor at high stringency all along the length of the nucleic acid ofinterest. Also contemplated are nucleic acid molecules that containdegenerate codons in place of codons in the hybridizing nucleic acidmolecule.

Whether any two molecules have nucleotide sequences or amino acidsequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% “identical” or “homologous” can be determined using knowncomputer algorithms such as the “FASTA” program, using for example, thedefault parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci.USA 85:2444 (other programs include the GCG program package (Devereux,J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN,FASTA (Altschul, S. F., et al., J Mol Biol 215:403 (1990)); Guide toHuge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994,and Carrillo, H. and Lipton, D., (1988) SIAM J Applied Math 48:1073).For example, the BLAST function of the National Center for BiotechnologyInformation database can be used to determine identity. Othercommercially or publicly available programs include, DNAStar “MegAlign”program (Madison, Wis.) and the University of Wisconsin GeneticsComputer Group (UWG) “Gap” program (Madison Wis.). Percent homology oridentity of proteins and/or nucleic acid molecules can be determined,for example, by comparing sequence information using a GAP computerprogram (e.g., Needleman et al. (1970) J. Mol. Biol. 48:443, as revisedby Smith and Waterman ((1981) Adv. Appl. Math. 2:482). Briefly, the GAPprogram defines similarity as the number of aligned symbols (i.e.,nucleotides or amino acids), which are similar, divided by the totalnumber of symbols in the shorter of the two sequences. Defaultparameters for the GAP program can include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)and the weighted comparison matrix of Gribskov et al. (1986) Nucl. AcidsRes. 14:6745, as described by Schwartz and Dayhoff, eds., ATLAS OFPROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation,pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

Therefore, as used herein, the term “identity” or “homology” representsa comparison between a test and a reference polypeptide orpolynucleotide. As used herein, the term at least “90% identical to”refers to percent identities from 90 to 99.99 relative to the referencenucleic acid or amino acid sequence of the polypeptide. Identity at alevel of 90% or more is indicative of the fact that, assuming forexemplification purposes a test and reference polypeptide length of 100amino acids are compared. No more than 10% (i.e., 10 out of 100) of theamino acids in the test polypeptide differs from that of the referencepolypeptide. Similar comparisons can be made between test and referencepolynucleotides. Such differences can be represented as point mutationsrandomly distributed over the entire length of a polypeptide or they canbe clustered in one or more locations of varying length up to themaximum allowable, e.g. 10/100 amino acid difference (approximately 90%identity). Differences are defined as nucleic acid or amino acidsubstitutions, insertions or deletions. At the level of homologies oridentities above about 85-90%, the result should be independent of theprogram and gap parameters set; such high levels of identity can beassessed readily, often by manual alignment without relying on software.

As used herein, an aligned sequence refers to the use of homology(similarity and/or identity) to align corresponding positions in asequence of nucleotides or amino acids. Typically, two or more sequencesthat are related by 50% or more identity are aligned. An aligned set ofsequences refers to 2 or more sequences that are aligned atcorresponding positions and can include aligning sequences derived fromRNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.

As used herein, “primer” refers to a nucleic acid molecule that can actas a point of initiation of template-directed DNA synthesis underappropriate conditions (e.g., in the presence of four differentnucleoside triphosphates and a polymerization agent, such as DNApolymerase, RNA polymerase or reverse transcriptase) in an appropriatebuffer and at a suitable temperature. It will be appreciated thatcertain nucleic acid molecules can serve as a “probe” and as a “primer.”A primer, however, has a 3′ hydroxyl group for extension. A primer canbe used in a variety of methods, including, for example, polymerasechain reaction (PCR), reverse-transcriptase (RT)-PCR, RNA PCR, LCR,multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3′ and 5′RACE, in situ PCR, ligation-mediated PCR and other amplificationprotocols.

As used herein, “primer pair” refers to a set of primers that includes a5′ (upstream) primer that hybridizes with the complement of the 5′ endof a sequence to be amplified (e.g. by PCR) and a 3′ (downstream) primerthat hybridizes with the 3′ end of the sequence to be amplified.

As used herein, “specifically hybridizes” refers to annealing, bycomplementary base-pairing, of a nucleic acid molecule (e.g. anoligonucleotide) to a target nucleic acid molecule. Those of skill inthe art are familiar with in vitro and in vivo parameters that affectspecific hybridization, such as length and composition of the particularmolecule. Parameters particularly relevant to in vitro hybridizationfurther include annealing and washing temperature, buffer compositionand salt concentration. Exemplary washing conditions for removingnon-specifically bound nucleic acid molecules at high stringency are0.1×SSPE, 0.1% SDS, 65° C., and at medium stringency are 0.2×SSPE, 0.1%SDS, 50° C. Equivalent stringency conditions are known in the art. Theskilled person can readily adjust these parameters to achieve specifichybridization of a nucleic acid molecule to a target nucleic acidmolecule appropriate for a particular application. Complementary, whenreferring to two nucleotide sequences, means that the two sequences ofnucleotides are capable of hybridizing, typically with less than 25%,15% or 5% mismatches between opposed nucleotides. If necessary, thepercentage of complementarity will be specified. Typically the twomolecules are selected such that they will hybridize under conditions ofhigh stringency.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, it also is understood that the terms “substantiallyidentical” or “similar” varies with the context as understood by thoseskilled in the relevant art.

As used herein, an allelic variant or allelic variation references anyof two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and can result in phenotypic polymorphism within populations. Genemutations can be silent (no change in the encoded polypeptide) or canencode polypeptides having altered amino acid sequence. The term“allelic variant” also is used herein to denote a protein encoded by anallelic variant of a gene. Typically the reference form of the geneencodes a wildtype form and/or predominant form of a polypeptide from apopulation or single reference member of a species. Typically, allelicvariants, which include variants between and among species typicallyhave at least 80%, 90% or greater amino acid identity with a wildtypeand/or predominant form from the same species; the degree of identitydepends upon the gene and whether comparison is interspecies orintraspecies. Generally, intraspecies allelic variants have at leastabout 80%, 85%, 90%, 95% or greater identity with a wildtype and/orpredominant form, including 96%, 97%, 98%, 99% or greater identity witha wildtype and/or predominant form of a polypeptide. Reference to anallelic variant herein generally refers to variations in proteins amongmembers of the same species.

As used herein, “allele,” which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for that gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide or several nucleotides, and caninclude modifications such as substitutions, deletions and insertions ofnucleotides. An allele of a gene also can be a form of a gene containinga mutation.

As used herein, species variants refer to variants in polypeptides amongdifferent species, including different mammalian species, such as mouseand human. For example for PH20, exemplary of species variants providedherein are primate PH20, such as, but not limited to, human, chimpanzee,macaque and cynomolgus monkey. Generally, species variants have 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or greatersequence identity. Corresponding residues between and among speciesvariants can be determined by comparing and aligning sequences tomaximize the number of matching nucleotides or residues, for example,such that identity between the sequences is equal to or greater than95%, equal to or greater than 96%, equal to or greater than 97%, equalto or greater than 98% or equal to greater than 99%. The position ofinterest is then given the number assigned in the reference nucleic acidmolecule. Alignment can be effected manually or by eye, particularly,where sequence identity is greater than 80%.

As used herein, a human protein is one encoded by a nucleic acidmolecule, such as DNA, present in the genome of a human, including allallelic variants and conservative variations thereof. A variant ormodification of a protein is a human protein if the modification isbased on the wildtype or prominent sequence of a human protein.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA.

As used herein, modification is in reference to modification of asequence of amino acids of a polypeptide or a sequence of nucleotides ina nucleic acid molecule and includes deletions, insertions, andreplacements (e.g. substitutions) of amino acids and nucleotides,respectively. Exemplary of modifications are amino acid substitutions.An amino-acid substituted polypeptide can exhibit 65%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity toa polypeptide not containing the amino acid substitutions. Amino acidsubstitutions can be conservative or non-conservative. Generally, anymodification to a polypeptide retains an activity of the polypeptide.Methods of modifying a polypeptide are routine to those of skill in theart, such as by using recombinant DNA methodologies.

As used herein, suitable conservative substitutions of amino acids areknown to those of skill in this art and can be made generally withoutaltering the biological activity of the resulting molecule. Those ofskill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. co., p. 224). Such substitutions can be made in accordance withthose set forth in TABLE 2 as follows:

TABLE 2 Exemplary conservative Original residue substitution Ala (A)Gly; Ser Arg ( R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E)Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; ValLys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser(S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu

Other substitutions also are permissible and can be determinedempirically or in accord with known conservative substitutions.

As used herein, the term promoter means a portion of a gene containingDNA sequences that provide for the binding of RNA polymerase andinitiation of transcription. Promoter sequences are commonly, but notalways, found in the 5′ non-coding region of genes.

As used herein, isolated or purified polypeptide or protein orbiologically-active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue fromwhich the protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. Preparationscan be determined to be substantially free if they appear free ofreadily detectable impurities as determined by standard methods ofanalysis, such as thin layer chromatography (TLC), gel electrophoresisand high performance liquid chromatography (HPLC), used by those ofskill in the art to assess such purity, or sufficiently pure such thatfurther purification would not detectably alter the physical andchemical properties, such as enzymatic and biological activities, of thesubstance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound, however, can be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

Hence, reference to a substantially purified polypeptide, such as asubstantially purified soluble PH20, refers to preparations of proteinsthat are substantially free of cellular material, which includespreparations of proteins in which the protein is separated from cellularcomponents of the cells from which it is isolated orrecombinantly-produced. In one embodiment, the term substantially freeof cellular material includes preparations of enzyme proteins havingless than about 30% (by dry weight) of non-enzyme proteins (alsoreferred to herein as a contaminating protein), generally less thanabout 20% of non-enzyme proteins or 10% of non-enzyme proteins or lessthan about 5% of non-enzyme proteins. When the enzyme protein isrecombinantly produced, it also is substantially free of culture medium,i.e., culture medium represents less than about or at 20%, 10% or 5% ofthe volume of the enzyme protein preparation.

As used herein, the term substantially free of chemical precursors orother chemicals includes preparations of enzyme proteins in which theprotein is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. The term includespreparations of enzyme proteins having less than about 30% (by dryweight), 20%, 10%, 5% or less of chemical precursors or non-enzymechemicals or components.

As used herein, synthetic, with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

As used herein, production by recombinant means or using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein, vector (or plasmid) refers to discrete elements that areused to introduce a heterologous nucleic acid into cells for eitherexpression or replication thereof. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Also contemplated are vectorsthat are artificial chromosomes, such as yeast artificial chromosomesand mammalian artificial chromosomes. Selection and use of such vehiclesare well known to those of skill in the art.

As used herein, an expression vector includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal. Expression vectors are generally derived fromplasmid or viral DNA, or can contain elements of both. Thus, anexpression vector refers to a recombinant DNA or RNA construct, such asa plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome.

As used herein, vector also includes “virus vectors” or “viral vectors.”Viral vectors are engineered viruses that are operatively linked toexogenous genes to transfer (as vehicles or shuttles) the exogenousgenes into cells.

As used herein, “operably” or “operatively linked” when referring to DNAsegments means that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiatesdownstream of the promoter and upstream of any transcribed sequences.The promoter is usually the domain to which the transcriptionalmachinery binds to initiate transcription and proceeds through thecoding segment to the terminator.

As used herein the term “assessing” is intended to include quantitativeand qualitative determination in the sense of obtaining an absolutevalue for the activity of a protein, such as an enzyme, or a domainthereof, present in the sample, and also of obtaining an index, ratio,percentage, visual or other value indicative of the level of theactivity. Assessment can be direct or indirect. For example, thechemical species actually detected need not of course be theenzymatically cleaved product itself but can for example be a derivativethereof or some further substance. For example, detection of a cleavageproduct can be a detectable moiety such as a fluorescent moiety.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Biologicalactivities can be observed in in vitro systems designed to test or usesuch activities. Thus, for purposes herein a biological activity of ahyaluronidase enzyme is its degradation of hyaluronic acid.

As used herein equivalent, when referring to two sequences of nucleicacids, means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When equivalent is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with only aminoacid substitutions that do not substantially alter the activity orfunction of the protein or peptide. When equivalent refers to aproperty, the property does not need to be present to the same extent(e.g., two peptides can exhibit different rates of the same type ofenzymatic activity), but the activities are usually substantially thesame.

As used herein, “modulate” and “modulation” or “alter” refer to a changeof an activity of a molecule, such as a protein. Exemplary activitiesinclude, but are not limited to, biological activities, such as signaltransduction. Modulation can include an increase in the activity (i.e.,up-regulation or agonist activity), a decrease in activity (i.e.,down-regulation or inhibition) or any other alteration in an activity(such as a change in periodicity, frequency, duration, kinetics or otherparameter). Modulation can be context dependent and typically modulationis compared to a designated state, for example, the wildtype protein,the protein in a constitutive state, or the protein as expressed in adesignated cell type or condition.

As used herein, a composition refers to any mixture. It can be asolution, suspension, liquid, powder, paste, aqueous, non-aqueous or anycombination thereof.

As used herein, a combination refers to any association between or amongtwo or more items. The combination can be two or more separate items,such as two compositions or two collections, can be a mixture thereof,such as a single mixture of the two or more items, or any variationthereof. The elements of a combination are generally functionallyassociated or related. For example, a combination can be a combinationof compositions provided herein.

As used herein a kit refers to a combination of components, such as acombination of the compositions herein and another item for a purposeincluding, but not limited to, reconstitution, activation, andinstruments/devices for delivery, administration, diagnosis, andassessment of a biological activity or property. Kits optionally includeinstructions for use.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions, andcharacterized by identifiable symptoms. Diseases and disorders ofinterest herein are hyaluronan-associated diseases and disorders.

As used herein, “treating” a subject with a disease or condition meansthat the subject's symptoms are partially or totally alleviated, orremain static following treatment. Hence treatment encompassesprophylaxis, therapy and/or cure. Prophylaxis refers to prevention of apotential disease and/or a prevention of worsening of symptoms orprogression of a disease.

As used herein, a pharmaceutically effective agent, includes anytherapeutic agent or bioactive agents, including, but not limited to,for example, chemotherapeutics, anesthetics, vasoconstrictors,dispersing agents, conventional therapeutic drugs, including smallmolecule drugs and therapeutic proteins.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease or other indication, are ameliorated orotherwise beneficially altered.

As used herein, therapeutic effect means an effect resulting fromtreatment of a subject that alters, typically improves or amelioratesthe symptoms of a disease or condition or that cures a disease orcondition. A therapeutically effective amount refers to the amount of acomposition, molecule or compound which results in a therapeutic effectfollowing administration to a subject.

As used herein, the term “subject” refers to an animal, including amammal, such as a human being.

As used herein, a “patient” refers to a human subject exhibitingsymptoms of a disease or disorder.

As used herein, an “individual” can be a subject.

As used herein, about the same means within an amount that one of skillin the art would consider to be the same or to be within an acceptablerange of error. For example, typically, for pharmaceutical compositions,an amount within at least 1%, 2%, 3%, 4%, 5% or 10% is considered aboutthe same. Such amount can vary depending upon the tolerance forvariation in the particular composition by subjects.

As used herein, dosing regime refers to the amount of agent, forexample, the composition containing a hyaluronan-degrading enzyme, forexample a soluble hyaluronidase or other agent, administered, and thefrequency of administration. The dosing regime is a function of thedisease or condition to be treated, and thus can vary.

As used herein, frequency of administration refers to the time betweensuccessive administrations of treatment. For example, frequency can bedays, weeks or months. For example, frequency can be more than onceweekly, for example, twice a week, three times a week, four times aweek, five times a week, six times a week or daily. Frequency also canbe one, two, three or four weeks. The particular frequency is functionof the particular disease or condition treated. Generally, frequency ismore than once weekly, and generally is twice weekly.

As used herein, a “cycle of administration” refers to the repeatedschedule of the dosing regime of administration of the enzyme and/or asecond agent that is repeated over successive administrations. Forexample, an exemplary cycle of administration is a 28 day cycle withadministration twice weekly for three weeks, followed by one-week ofdiscontinued dosing.

As used herein, when referencing dosage based on mg/kg of the subject,an average human subject is considered to have a mass of about 70 kg-75kg, such as 70 kg.

As used herein, amelioration of the symptoms of a particular disease ordisorder by a treatment, such as by administration of a pharmaceuticalcomposition or other therapeutic, refers to any lessening, whetherpermanent or temporary, lasting or transient, of the symptoms or,adverse effects of a condition, such as, for example, reduction ofadverse effects associated with or that occur upon administration of ahyaluronan-degrading enzyme, such as a PEGylated hyaluronidase.

As used herein, prevention or prophylaxis refers to a reduction in therisk of developing a disease or condition.

As used herein, a “therapeutically effective amount” or a“therapeutically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that is atleast sufficient to produce a therapeutic effect. Hence, it is thequantity necessary for preventing, curing, ameliorating, arresting orpartially arresting a symptom of a disease or disorder.

As used herein, unit dose form refers to physically discrete unitssuitable for human and animal subjects and packaged individually as isknown in the art.

As used herein, a single dosage formulation refers to a formulation as asingle dose.

As used herein, formulation for direct administration means that thecomposition does not require further dilution for administration.

As used herein, an “article of manufacture” is a product that is madeand sold. As used throughout this application, the term is intended toencompass anti-hyaluronan agents, for example hyaluronan-degradingenzyme, such as hyaluronidase, and second agent compositions containedin articles of packaging.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, a cellular extract or lysate refers to a preparation orfraction which is made from a lysed or disrupted cell.

As used herein, animal includes any animal, such as, but are not limitedto primates including humans, gorillas and monkeys; rodents, such asmice and rats; fowl, such as chickens; ruminants, such as goats, cows,deer, sheep; pigs and other animals. Non-human animals exclude humans asthe contemplated animal. The hyaluronidases provided herein are from anysource, animal, plant, prokaryotic and fungal. Most hyaluronidases areof animal origin, including mammalian origin. Generally hyaluronidasesare of human origin.

As used herein, anti-cancer treatments include administration of drugsand other agents for treating cancer, and also treatment protocols, suchas surgery and radiation. Anti-cancer treatments include administrationof anti-cancer agents.

As used herein, an anti-cancer agent or refers to any agents, orcompounds, used in anti-cancer treatment. These include any agents, whenused alone or in combination with other compounds, that can alleviate,reduce, ameliorate, prevent, or place or maintain in a state ofremission of clinical symptoms or diagnostic markers associated withtumors and cancer, and can be used in combinations and compositionsprovided herein. Exemplary anti-cancer agents include, but are notlimited to, hyaluronan-degrading enzymes, such as the PEGylatedhyaluronan-degrading enzymes provided herein used singly or incombination with other anti-cancer agents, such as chemotherapeutics,polypeptides, antibodies, peptides, small molecules or gene therapyvectors, viruses or DNA.

As used herein, a control refers to a sample that is substantiallyidentical to the test sample, except that it is not treated with a testparameter, or, if it is a plasma sample, it can be from a normalvolunteer not affected with the condition of interest. A control alsocan be an internal control.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a compound comprising or containing “anextracellular domain” includes compounds with one or a plurality ofextracellular domains.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 bases” means “about 5 bases” and also “5 bases.” Generally“about” includes an amount that would be expected to be withinexperimental error.

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally substitutedgroup means that the group is unsubstituted or is substituted.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).

B. HYALURONAN BINDING PROTEIN AND COMPANION DIAGNOSTIC

Provided herein are sensitive and specific methods to detect and closelymonitor hyaluronan (HA) levels associated with disease, particularly inthe extracellular matrix (ECM) of tumor tissues. The companiondiagnostic methods provided herein are based on the finding that HAaccumulation specifically correlates with and predicts aggressivedisease, in particular with respect to cancers. In addition, thecompanion diagnostic method provided herein also is based on the findingthat HA specifically provides superior prognostic and treatmentselection information as compared to other markers involved in the HAmetabolic pathway associated with hyaluronan-associated diseases andconditions, such as hyaluronidase synthases or hyaluronidases. Hence,the method provided herein utilizes improved hyaluronan binding protein(HABP) reagents that exhibit specificity, high affinity and lowvariability for specific and sensitive detection of HA. Also providedherein are improved HABP reagents.

In one example, the improved HABPs provided herein, such as anydescribed in Section C, can be a companion diagnostic for selectingpatients with HA-associated diseases, for example HA-associated tumors,for treatment with an anti-hyaluronan agent or hyaluronan-degradingenzyme, such as any set forth in Section E (e.g. a hyaluronidase ormodified hyaluronidase such as PEGylated PH20, i.e. PEGPH20). In such anexample, the method is useful in classification of patients forselection of therapy, such as cancer therapy, and in particular relatesto the measurement of HA levels that correlate with responsiveness totherapy with an anti-hyaluronan agent, for example ahyaluronan-degrading enzyme therapy, such as therapy by a PEGPH20 fortreatment of patients with advanced tumors.

In another example, the improved HABPs provided herein, such as anydescribed in Section C, also can be used in methods of monitoringefficacy or responsiveness to treatment with an anti-hyaluronan agent orhyaluronan-degrading enzyme such as any set forth in Section E (e.g. ahyaluronidase or modified hyaluronidase such as PEGylated PH20, i.e.PEGPH20) by detecting levels of HA during the treatment. Thus, theimproved HABP can be used in conjunction with therapy with ananti-hyaluronan-agent, for example hyaluronan-degrading enzyme therapy,to monitor HA levels and to adjust and/or alter therapy to personalizeindividual treatment of a patient depending upon the particular patientand course of disease in a manner that correlates to clinical response.

Also provided herein are combinations and kits that contain ananti-hyaluronan agent, such as a hyaluronan-degrading enzyme (forexample any provided herein below in Section E) and an improved HABP(for example any provided herein below in Section C), and optionallyother accompanying reagents, for use in selecting, monitoring and/ortreating HA-associated diseases and conditions, in particular cancer.

1. Hyaluronan Accumulation in Disease and Correlation to Prognosis

Hyaluronan (HA; also called hyaluronic acid or hyaluronate) is a linearglycosaminoglycan (GAG) polymer containing repeating N-acetylglucosamineand D-glucuronic acid disaccharide subunits viaGlcUA-β1,3-GlcNAc-β1,4-linkages. Hyaluronan is synthesized by a class ofhyaluronan synthases, HAS1, HAS2 and HAS3. These enzymes act bylengthening hyaluronan by adding glucuronic and N-acetylglucosamine tothe nascent polysaccharide as it is extruded through the cell. Inaddition to the HA synthases, the level of HA normally is maintained byits catabolism by hyaluronidases, specifically the turnover enzymehyaluronidase 1 (Hyal1). The dynamic turnover of HA is balanced bybiosynthesis and catabolism to keep a constant concentration in thenormal tissue.

HA is a component of the extracellular matrix (ECM). It is ubiquitouslydistributed in tissues and localized in the extracellular, pericellularmatrices as well as inside cells. HA has a wide range of biologicalfunctions such as contributing to tissue homeostasis and biomechanics,cell proliferation, immune adhesion and activation, and cell migrationduring dynamic cellular processes. These processes are mediated byinteraction of HA with HA-binding proteins known as hyaladherins, suchas TSG-6, versican, inter-alpha-trypsin inhibitor, CD44, lymphaticvessel endothelial HA receptor (LYVE-1-1) and RHAMM.

Hyaluronan accumulation is associated with many malignant and autoimmunedisease conditions (Järveläinen H, et al. (2009) Pharmacol Rev 61:198-223; Whatcott C J, et al. (2011) Cancer Discovery 1:291-296). Forexample, certain diseases are associated with expression and/orproduction of hyaluronan, including inflammatory diseases and cancers.HA is linked to a variety of biological processes involved withprogression of such diseases (see e.g. Itano et al. (2008) Semin CancerBiol 18(4):268-274; Tammi et al. (2008) Semin Cancer Biol18(4):288-295).

In particular, HA is a component of the tumor matrix and is present inmany solid tumors. Accumulation of HA within a tumor focus preventscell-cell contact, promotes epithelial-mesenchymal transitions, isinvolved with the p53 tumor suppressor pathway via its receptors RHAMMand CD44 and recruits tumor-associated macrophages (Itano et al. (2008)Cancer Sci 99: 1720-1725; Camenisch et al. (2000) J Clin Invest106:349-360; Thompson et al. (2010) Mol. Cancer Ther. 9:3052-64). Theassembly of a pericellular matrix rich in HA is a prerequisite forproliferation and migration of mesenchymal cells that can promotemetastatic behavior. Tumors characterized by the accumulation of HA alsoexhibit tumor water uptake and have high interstitial fluid pressure(IFP) that can inhibit penetration of and accessibility of the tumor tosystemically applied therapeutics, such as chemotherapeutics. Further,HA oligomers, generated by degradation by Hyal1, also have been shown toresult in angiogenesis or apoptosis that can contribute to tumorpathogenesis.

The accumulation of HA has been correlated to HAS gene expression and/orHYAL gene expression (Kosaki et al. (1999) Cancer Res. 59:1141-1145; Liuet al. (2001) Cancer Res. 61:5207-5214; Wang et al. (2008) PLoS 3:3032;Nykopp et al. (2010) BMC Cancer 10:512). Studies in the art havevariously shown that HA, HAS or Hyal1 can be used as prognosticindicators of cancer. Also, studies have suggested that the selectiveinhibition of Hyal1, such as by anti-sense methods, or of hyaluronansynthesis by HAS, such as by the use of 4-methylumbelliferone, aremethods of treating tumors (Kakizaki et al. (2004) J. Biol. Chem.279:33281-33289). In addition, hyaluronidases, such as PH20 as discussedbelow, also have been used to treat hyaluronan-associated diseases andconditions (see e.g. Thompson et al. (2010) Mol. Cancer Ther9:3052-3064).

As shown in the Examples, it is now found herein that the HA phenotypeof a cell, and in particular the formation of a tumor pericellularmatrix, correlates to tumor aggressiveness, and that an assay for HAlevels specifically predicts that ability of HA-synthesizing tumorscells to form a pericellular matrix. Specifically, it is found hereinthat among the potential markers of HA accumulation, including HAS1, 2,3; Hyal1 or 2; or HA, that only HA determination correlated withpericellular matrix formation and thereby predicted tumor cellcompetence to form HA-aggrecan-mediated pericellular matrices andthereby tumor aggressiveness. Thus, for purposes of a diagnostic topredict or prognose tumor therapy, an HA binding protein (HABP) iscontemplated. As described herein, tumor HA production can be measuredquantitatively using an HABP as a probe, and HABP for hyaluronan shows acorrelation to pericellular matrix formation while no correlation wasfound between pericellular matrix formation and relative levels of HASor Hyal mRNA. These findings show that direct measurement of tumorcell-associated HA, and not the other markers involved in the HAmetabolic pathway, offers a reliable predictor for pericellular matrixformation.

2. Therapy of Tumors with an Anti-Hyaluronan Agent (e.g.Hyaluronan-Degrading Enzyme) and Responsiveness to Treatment

It also is found herein that the amount or extent of HA accumulationmeasured also correlates with responsiveness to treatment with ananti-hyaluronan agent, for example a hyaluronan-degrading enzyme, suchas PH20. Anti-Hyaluronan agents, for example hyaluronan-degradingenzymes, such as a PH20, exhibit properties useful for single-agent orcombination therapy of diseases and conditions that exhibit theaccumulation of hyaluronan (hyaluronic acid, HA). Suchhyaluronan-associated diseases, conditions and/or disorders includecancers and inflammatory diseases. Hyaluronan-rich cancers include, butare not limited to, tumors, including solid tumors, for example,late-stage cancers, a metastatic cancers, undifferentiated cancers,ovarian cancer, in situ carcinoma (ISC), squamous cell carcinoma (SCC),prostate cancer, pancreatic cancer, non-small cell lung cancer, breastcancer, colon cancer and other cancers.

For example, HA degrading enzymes, such a hyaluronidase, for examplePH20, have been shown to remove HA from tumors resulting in thereduction of tumor volume, the reduction of IFP, the slowing of tumorcell proliferation, and the enhanced efficacy of co-administeredchemotherapeutic drugs and biological agents by permitting increasedtumor penetration (see e.g. U.S. published application No. 20100003238and International published PCT Appl. No WO 2009/128917; Thompson et al.(2010) Mol. Cancer Ther 9:3052-3064).

The ability of a hyaluronidase, such as PH20, to degrade HA to serve asa therapeutic of hyaluronan-associated diseases and disorders can beexploited by modification to increase systemic half-life. The increasedhalf-life permits not only removal of HA, but also, due to its continuedpresence in the plasma and its ability to degrade HA, reduces ordecreases the extent of regeneration of HA within diseased tissues, suchas the tumor. Hence, maintenance of plasma enzyme levels can remove HA,such as tumor HA, and also counteract HA resynthesis. PEGylation is anestablished technology used to increase the half-life of therapeuticproteins in the body thus enabling their use in systemic treatmentprotocols. PEGylation of anti-hyaluronan agents, such ashyaluronan-degrading enzymes, such as hyaluronidase extends itshalf-life in the body from less than a minute to approximately 48 to 72hours and allows for the systemic treatment of tumors rich in HA (seee.g. U.S. published application No. 20100003238 and Internationalpublished PCT Appl. No WO 2009/128917; Thompson et al. (2010) Mol CancerTher 9: 3052-3064).

It is found herein that the growth inhibitory activity of ananti-hyaluronan agent, and in particular a hyaluronan-degrading enzyme,for example a hyaluronidase, such as a PH20 or PEGPH20, on tumor cellsis correlated with the extent of HA levels. As shown in the Examples,tumors can be characterized into phenotypic groups (e.g. HA⁺¹, HA⁺²,HA⁺³) based on the amount of HA expression in the tumor. Hightumor-associated HA (scored HA⁺³) resulted in accelerated tumor growthin animal models and to greater tumor inhibition by ahyaluronan-degrading enzyme (e.g. PEGPH20). For example, tumor growthinhibition associated with an HA⁺³ phenotype was 97%, whereas it wasonly 44% and 16% for tumor HA⁺² or HA⁺¹ phenotypes, respectively. Thedata indicate the continued growth of some tumors is dependent upon thedensity and amount of HA in the tumor microenvironment and thatdepletion of HA from an HA rich (e.g. HA⁺³) tumor has a more pronouncedeffect on tumor growth than depletion of HA from an HA moderate or poortumor (e.g., HA⁺², HA⁺¹) or HA deficient tumor. Thus, as shown herein,the degree of HA accumulation in tumor tissues, as measured using anHABP, is predictive of the level of inhibition of tumor growth in vivomediated by an anti-hyaluronan agent (e.g., PEGPH20).

3. Hyaluronan Binding Proteins (HABPs) Reagent and Diagnostic

Based on the results provided above and in the Examples herein, thebiomarker HA detected using an HABP has been specifically correlated toresponse to an anti-hyaluronan treatment, for example ahyaluronan-degrading enzyme treatment (e.g. PEGPH20). Thus, providedherein is a method of using an HABP for prognosis and also to predictthe degree of sensitivity, and thus responsiveness, to ananti-Hyaluronan agent, for example a hyaluronan-degrading enzyme (e.g. ahyaluronidase or modified hyaluronidase such as PEGylated PH20, i.e.PEGPH20).

For value as a reagent, the sensitivity and specificity of an HABP isdesired as well as reproducibility due to low variability. For example,the detection and measurement of HA in tissues is limited using existingreagents. Currently, the method used to detect or measure HA in tissuesvia immunohistological staining is mainly dependent on the animalcartilage tissue-derived HA binding proteins or domains. These includethe HABP purified from bovine nasal cartilage proteoglycan by extractionwith 4 M guanidine HCl and then by affinity chromatography using HAcoupled resin. The resulting animal-derived HA is composed of two majorcomponents: Aggrecan G1 domain and link module. Due to variation frombatch to batch, as well as different modifications used in the method toprepare the HABP, variability exists in the art in terms of differencesin the HABP staining patterns and the discrepancies in staining profilesmake comparisons among studies difficult. Thus, due to its existence asa heterogenous mixture of components and no validated procedure for itsproduction, alternative HABP proteins are provided herein for use incompanion diagnostics for prognosis of disease and predicting efficacyof treatment in conjunction with an anti-hyaluronan agent (e.g. ahyaluronan-degrading enzyme).

Hence, the HABP reagent for use in the methods provided herein includesany HABP that is not an HABP purified from animal cartilage, forexample, purified from bone nasal cartilage as used in the art using amethod as described by E-Laurent et al. (1985) Ann. Rheum. Dis.44:83-88) or modified method thereof. Exemplary HABPs for use in themethods herein are described in Section C. Such proteins include, forexample, HABPs containing one or more HA binding domains, including oneor more link modules for binding to HA. In some examples, the HABPcontains an HA binding domain (e.g. a link module) of aggrecan,versican, neurocan, brevican, phosphacan, HAPLN-1, HAPLN-2, HAPLN-3,HAPLN-4, stabilin-1, stabilin-2, CAB61358, KIAA0527, or TSG-6 protein.In some examples, the HABP contains an aggrecan G1 domain, versican G1domain, neurocan G1 domain, brevican G1 domain, or a phosphacan G1domain. In some examples, the HABP contains a G1 domain of aggrecan,versican, neurocan, brevican, or phosphacan and a link protein selectedfrom HAPLN-1, HAPLN-2, HAPLN-3, or HAPLN-4. In some examples, the HABPcontains a link module of TSG-6, stabilin-1, stabilin-2, CAB61358, orKIAA0527.

In some examples, the HABP is a modified HABP, such as, for example amodified aggrecan, versican, neurocan, brevican, phosphacan, HAPLN-1,HAPLN-2, HAPLN-3, HAPLN-4, stabilin-1, stabilin-2, CAB61358, KIAA0527,or TSG-6 protein, such as, for example TSG-6-LM-Fc. In some examples,the HABP is a modified HABP that is modified to improve its binding toHA, such as, for example, TSG-6-LM-FcΔHep.

In particular, the HABP provided herein 1) can be produced recombinantlyin an expression system, such as a mammalian expression system; 2)exhibits improved biophysical properties such as stability and/orsolubility; 3) can be purified by simple purification methods, such asby one-step affinity purification methods; 4) is capable of beingdetected by procedures compatible with binding assays, and in particularimmunohistochemistry or ELISA methods; 5) can be expressed in multimericform (e.g. via dimerization) to exhibit increased or high affinity forHA; and/or 6) exhibits specificity for HA as compared to other GAGs.

In one example, provided herein for use in the methods herein are HABPsthat are single module HA proteins that can be produced recombinantly inexpression systems. In particular, provided herein are HABP reagentsthat contain a link module. For example, HABPs provided herein are ofthe type A class of HABPs containing only the link module (LM) or asufficient portion thereof to bind to hyaluronan. Exemplary of suchHABPs are tumor necrosis factor-stimulated Gene (TSG)-6-LM (link moduleset forth in SEQ ID NO:360), stabilin-1-LM or stabilin-2-LM (link moduleset forth in SEQ ID NO:371 or 372, respectively), CAB61358-LM (linkmodule set forth in SEQ ID NO: 373) or KIAA0527-LM (link module setforth in SEQ ID NO:374).

In another example, provided herein for use in the methods herein areHABPs that are linked directly or indirectly to a multimerizationdomain. HA-binding domains, such as a link module, of HABPs can bedirectly or indirectly linked, such as covalently-linked,non-covalently-linked or chemically linked, to form multimers of two ormore HA binding domains. The HA binding domains can be the same ordifferent. In particular, the HA binding domain is a link domain ormodule. Hence, multimers can be formed by dimerization of two or morelink domains. In one example, multimers can be linked by disulfide bondsformed between cysteine residues on different HA-link domains. Forexample, a multimerization domain can include a portion of animmunoglobulin molecule, such as a portion of an immunoglobulin constantregion (Fc). In another example, multimers can include an HA-bindingdomain joined via covalent or non-covalent interactions to peptidemoieties fused to the polypeptide. Such peptides can be peptide linkers(spacers), or peptides that have the property of promotingmultimerization. In additional example, multimers can be formed betweentwo polypeptides through chemical linkage, such as for example, by usingheterobifunctional linkers. A description of multimerization domains isprovided below. Exemplary of an HABP multimer is a link module (LM)fused to an Fc. For example, exemplary of an HABP reagent for use in themethods herein is TSG-6-LM-Fc.

In a further example, provided herein for use in the methods herein areHABP that are modified, such as by amino acid replacement, to exhibitincreased specificity for hyaluronan compared to other GAGs. Forexample, provided herein is a mutant TSG-6-LM containing amino acidreplacement(s) at amino acid residues 20, 34, 41, 54, 56, 72 and/or 84,and in particular at amino acid residues 20, 34, 41, and/or 54(corresponding to amino acid residues set forth in SEQ ID NO:206). Thereplacement amino acid can be to any other amino acid residue, andgenerally is to a non-basic amino acid residue. For example, amino acidreplacement can be to Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln(Q), Ala (A), Val (V), Ile (I), Leu (L), Met (M), Phe (F), Tyr (Y) orTrp (W). The amino acid replacement or replacements confer decreasedbinding to heparin. Binding can be reduced at least 1.2-fold, 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 100-fold or more compared to bindingof TSG-6-LM to heparin not containing the amino acid replacement.Exemplary of a TSG-6-LM mutant for use as a reagent in the methodprovided herein is K20A/K34A/K41A. Hence, for example, binding toheparin is reduced such that specificity to hyaluronan is increased. Themutant TSG-6-LM can be conjugated directly or indirectly to amultimerization domain to generate multimers. For example, exemplary ofa reagent for use in the methods herein is TSG-6-LM(K20A/K34A/K41A)-Fc.

Any of the reagents can be used alone or in combination with a companiondiagnostic method. For example, in a sandwich ELISA or competitiveELISA, two or more of the above reagents can be used. As describedherein below, any of the HABPs provided herein can be conjugateddirectly or indirectly to a moiety that is capable of detection. In someexamples, the HABPs that bind to HA, for example in a tumor sample, canbe detected using a secondary reagent, such as an antibody that binds tothe HABP. In some examples, the HABPs are modified to permit detectionof HA binding. For example, the HABPs can be conjugated to a detectablemolecule that permits either direct detection or detection via secondaryagents, such as antibodies that bind to the modified HABPs and arecoupled to detectable proteins, such as fluorescent probes or detectableenzymes, such as horseradish peroxidase.

4. Companion Diagnostic and Prognostic Methods

The HABPs provided herein can be used either individually or incombination with diagnostic, prognostic or monitoring methods utilizingbinding assays on various biological samples of patients having ahyaluronan-associated disease or condition or at risk or suspected ofhaving a hyaluronan-associated disease or condition. For example, theHABPs can be used in assays on patients having a solid tumor or at riskof developing a solid tumor or other cancer. In particular examples, aTSG-6-LM, TSG-6-LM-Fc or variant or mutant thereof such as one thatexhibits reduced binding to heparin and increased specificity forhyaluronan is used in the methods herein. The diagnostic and prognosticmethods can be used in conjunction with therapy with ahyaluronan-degrading enzyme in order to classify and/or select patientsfor treatment or to alter or modify the course of treatment.

In exemplary methods provided herein, the diagnostic and prognosticmethods are companion methods to therapy with an anti-hyaluronan agent,such as a hyaluronan-degrading enzyme, for example a hyaluronidase ormodified hyaluronidase such as PH20 or PEGPH20. HA detection can informtreatment selection, initiation, dose customization or termination, andthus can serve to individualize treatment with an anti-hyaluronan agent,for example a hyaluronan-degrading enzyme.

For example, an HABP companion diagnostic method can be used todetermine whether a subject who is predisposed to ahyaluronan-associated disease or condition (e.g. cancer) or who issuffering from a hyaluronan-associated disease or condition (e.g.cancer) will benefit from or is predicted to be responsive to receivingtreatment with an anti-hyaluronan agent, such as a hyaluronan-degradingenzyme. In the method, the level of HA expression from samples fromsubjects predisposed or known to have a hyaluronan-associated disease orcondition (e.g. cancer) can be determined and the level of HA expressionin samples from subjects compared to predetermined HA levels thatclassify responsiveness to an anti-hyaluronan agent, for example ahyaluronan-degrading enzyme. It is within the level of one of skill inthe art to determine the threshold level of HA for classifyingresponsiveness to treatment with a hyaluronan-degrading enzyme. Forexample, it is found herein that a significant correlation existsbetween elevated HA accumulation and tumor growth inhibition, wherebytumor growth inhibition response correlated to an HA⁺³ phenotype asquantified by immunohistochemistry of tumor tissue. Thus, in thecompanion diagnostic method provided herein, a tumor sample is assessedfor HA levels using an HABP reagent provided herein byimmunohistochemistry methods or other methods adaptable to scoring. Ifthe HA phenotype is HA⁺³ as determined by methods known to one of skillin the art and described herein, then the subject is selected as acandidate for treatment with a hyaluronan-degrading enzyme, such as ahyaluronidase or modified hyaluronidase (e.g. PH20 or PEGPH20). Similarquantification and classification methods can be utilized by assessingHA in bodily fluids, such as blood or plasma. Dosages and regimens of ahyaluronan-degrading enzyme, such as PH20 or PEGPH20, for treatment areprovided herein.

The HABP reagents provided herein can detect HA using any binding assayknown to one of skill in the art including, but not limited to, enzymelinked immunosorbent assay (ELISA) or other similar immunoassay,including a sandwich ELISA or competitive ELISA assay;immunohistochemistry (IHC); flow cytometry, or western blot. The bindingassay can be performed on samples obtained from a patient body fluid,cell or tissue sample of any type, including from plasma, urine, tumoror suspected tumor tissues (including fresh, frozen, and fixed orparaffin embedded tissue), lymph node tissue or bone marrow.

Once the amount of HA in the sample is determined, the amount can becompared to a control or threshold level. For example, if the amount ofHA is determined to be elevated in the sample, the subject is selectedas a candidate for tumor therapy. Exemplary methods for stratificationof tumor samples or bodily fluid samples for diagnosis, prognosis orselection of subjects for treatment are provided herein.

In one example, a method of diagnosis utilizes a sample of tumor tissue,tumor cells or a bodily fluid containing proteins from a patient. In themethod, the presence and level of expression of HA can be determinedusing an HABP, for example a TSG-6-LM, TSG-6-LM-Fc or variant or mutantthereof, as provided herein. The level of expression of the HA isdetermined and/or scored and compared to predetermined HA phenotypesassociated with disease. As described below, these predetermined valuescan be determined by comparison or knowledge of HA levels in acorresponding normal sample as determined by the same assay of detectionand using the same HABP reagent. It is within the level of one of skillin the art to determine the threshold level for disease diagnosisdepending on the particular disease, the assay being used for detectionof HA and/or the HABP detection reagent being used. For example, inbodily fluids such as plasma, HA levels greater than 0.015 μg/mL, andgenerally greater than 0.02 μg/mL, 0.03 μg/mL, 0.04 μg/mL, 0.05 μg/mL,0.06 μg/mL or higher correlates to the presence of a tumor or cancer. Inanother example, in immunohistochemistry methods of tumor tissues with ascore of HA⁺² or HA⁺³ can be determinative of disease. If the level isindicative of disease, then the patient is diagnosed with having atumor.

In another example, a prognostic method utilizes a sample of tumortissue, tumor cells or a bodily fluid containing proteins from apatient. In the method, the presence and level of expression of HA canbe determined using an HABP, for example a TSG-6-LM, TSG-6-LM-Fc orvariant or mutant thereof, as provided herein. The level of expressionof the HA is determined and/or scored and compared to predetermined HAphenotypes associated with disease. As described below, thesepredetermined values can be determined by comparison or knowledge of HAlevels in a corresponding normal sample or samples of disease subjectsas determined by the same assay of detection and using the same HABPreagent. It is within the level of one of skill in the art to determinethe threshold levels for prognosis of disease depending on theparticular disease, the assay being used for detection of HA and/or theHABP detection reagent being used. The level of expression of HAindicates the expected course of disease progression in the patient. Forexample, high levels of HA as assessed by immunohistochemistry methodsusing a quantitative score scheme (e.g. HA⁺³) correlate to the existenceof malignant disease across a range of cancer types. In another example,HA levels in bodily fluid such as plasma of greater than 0.06 μg/mL HAalso is associated with advanced disease stage.

In a further example of companion diagnostic methods, the level of HAexpression in samples from subjects previously treated with ahyaluronan-degrading enzyme can be monitored to determine whether asubject being administered the agent has obtained an efficacious bloodlevel of the drug in order to optimize dosing or scheduling.

The following sections describe exemplary HABP reagents and assays forperforming the HA detection methods for use in the diagnostic andprognostic methods, and in particular as companions to therapy with ananti-hyaluronan agent, for example a hyaluronan-degrading enzyme. Alsodescribed are anti-hyaluronan agents, including hyaluronan-degradingenzyme agents, for use in treating hyaluronan-associated diseases anddisorders and kits and combinations of HABP reagents with such agents(e.g. hyaluronan-degrading enzymes). Any of the above methods can beperformed using any of the described HABP reagents and assay detectionmethods alone or in conjunction with therapy with an anti-hyaluronanagent (e.g. a hyaluronan-degrading enzyme).

C. HYALURONAN BINDING PROTEINS (HABPS) FOR USE AS A COMPANION DIAGNOSTIC

The methods provided herein are directed to quantitative orsemi-quantitative measurement of hyaluronan in a sample, such as a tumoror fluid sample from a subject having a tumor or suspected of having atumor, using a hyaluronan binding protein (HABP). As described herein,tumors that express elevated or high levels of hyaluronan are responsiveto treatment with an anti-hyaluronan agent (e.g. hyaluronan degradingenzyme) and the degree of tumor inhibition by an anti-hyaluronan agent(e.g. hyaluronan degrading enzyme) correlates with the degree or amountof hyaluronan accumulation, and not other markers such as expression ofendogenous hyaluronan synthases or hyaluronidases. The HABPs providedfor use in the methods herein, in concert with the assays for detectionthereof described in Section D, permit specific and sensitive detectionof HA in samples.

The HABP companion diagnostics provided herein can be used inconjunction with therapy with an anti-hyaluronan agent, such ashyaluronan-degrading enzyme therapeutics or any described in Section E,to select or identify patients predicted to be responsive to treatmentand/or to monitor treatment and efficacy of treatment, thereby providingan improved treatment regimen of hyaluronan-associated diseases orconditions. For example, the HABP companion diagnostics provided hereincan be used to select and/or monitor subjects or patients having a tumoror cancer. In addition, the HABP companion diagnostics also can be usedin other diagnostic and prognostic methods of hyaluronan-associateddisease or condition, such as tumors or cancers.

Provided herein are hyaluronan binding proteins for use in the methodsprovided herein for the detection and quantitation of hyaluronan in asample. The hyaluronan binding proteins can contain full length HABPpolypeptides, or portions thereof containing an HA binding domains ofHABPs, or sufficient portions thereof to bind HA. Typically, the HABPsor portions thereof containing an HA binding domain or sufficientportion thereof that binds HA, or variants or multimers thereof exhibita binding affinity with a dissociation constant (Kd) of at least lessthan or less than or 1×10⁻⁷ M, and generally at least less than or lessthan or 9×10⁻⁸ M, 8×10⁻⁸ M, 7×10⁻⁸ M, 6×10⁻⁸ M, 5×10⁻⁸ M, 4×10⁻⁸ M,3×10⁻⁸ M, 2×10⁻⁸ M, 1×10⁻⁸ M, 9×10⁻⁹ M, 8×10⁻⁹ M, 7×10⁻⁹ M, 6×10⁻⁹ M,5×10⁻⁹ M, 4×10⁻⁹ M, 3×10⁻⁹ M, 2×10⁻⁹ M, 1×10⁻⁹ M or lower Kd. Asdiscussed herein, the exhibited binding affinity is generally exhibitedunder conditions that achieve optimal or close to optimal binding tohyaluronan. In one example, pH conditions can affect binding. Forexample, as a companion diagnostic herein, binding assays using a TSG-6reagent, including TSG-6-LM or sufficient portions thereof to bind HA,variants thereof and multimers thereof, are generally conducted at a pHof at or about between pH 5.8 to 6.4, such as about or pH 6.0.

Hyaluronan binding proteins are of two types: hyaluronan bindingproteins that have an HA binding domain that contains one or two linkmodules, and hyaluronan binding proteins that have an HA binding domainthat is not a link module. In particular examples, the companiondiagnostics provided herein are derived from HABP binding molecules thathave only a single link domain that confers HA binding, which cansimplify expression, production and purification methods.

The HABPs provided herein can be derived from known HABPs or can begenerated synthetically. In some examples, HABPs can be generatedsynthetically based on conserved residues of HA-binding domains of knownHABPs. HABPs provided herein also can be derived from HABPs generatedfrom screening methods for HA binding proteins, such as phage display oraffinity-based screening methods.

The HABPs, including HA binding domains of HABPs, or portions thereofthat are sufficient to bind to HA, provided herein can be modified toimprove one or more properties of HABPs for use in the methods providedherein. For example, the HABPs, or HA binding fragments thereof,provided herein can be modified to increase protein expression inmammalian expression systems, improve biophysical properties such asstability and solubility, improve protein purification and detection,increase specificity for HA and/or increase affinity to HA, as long asthey retain their ability to bind to HA. For example, an HABP or HAbinding fragment thereof provided herein for use in the methods can bemodified to increase its specificity for hyaluronan compared to otherglycosaminoglycans. In another example, an HABP or HA binding fragmentthereof provided herein for use in the methods can be linked directly orindirectly to a multimerization domain to increase the number of HAbinding sites on the molecule and therefore increase the affinity forbinding to HA.

Further, for use as a companion diagnostic herein, any of the HABPs, orportions thereof (e.g. link modules or sufficient portions thereof tobind HA) can be modified to facilitate detection. For example, thecompanion diagnostics are modified by conjugation, directly orindirectly, to biotin, a fluorescent moiety, a radiolabel or otherdetectable label.

A description of exemplary HABPs for use as companion diagnosticsherein, and modifications thereof, is provided below.

1. HA Binding Proteins with Link Modules or G1 Domains

Provided herein as companion diagnostic reagents for use in the methodsherein are HA binding proteins (HABP) or portions thereof that containat least one link module or link domain, and generally at least two ormore link modules. In some examples, the HABP contains a G1 domain thatcontains two link modules. Binding to HA is mediated via the linkmodule. Link modules, also called proteoglycan tandem repeats, areapproximately 100 amino acids (aa) in length with four cysteines thatare disulfide bonded in the pattern Cys1-Cys4 and Cys2-Cys3. The threedimensional structure of the link modules are composed of twoalpha-helices and two triple stranded anti-parallel beta-sheets.

There are three categories of link module-containing proteins: Adomain-type proteins that contain a single link module; B domain-typeproteins that contain a single link module extended by an N- and aC-terminal flanking region; and C domain-type proteins that have anextended structure called a G1 domain that contains one N-terminalV-type Ig-like domain followed by a contiguous pair of two link modules.Modeling and comparison studies have demonstrated a high degree ofresolution and conservation of certain amino acids between and amonglink module-containing proteins that correlate to interaction with HA(Blundell et al. (2005) J. Biol. Chem., 280:18189-18201). For example,central HA-binding amino acid residues corresponding to Tyr59 and Tyr78with numbering with reference to TSG-6-LM set forth in SEQ ID NO:360 areconserved among link-module-containing HABPs via identical orconservative amino acids (e.g. aromatic or large and planar facedhydrophobic residues that can also stack against a GlcNAc ring, e.g.,Phe, His, Leu or Val) at the corresponding position based on alignmentwith TSG-6-LM (e.g. set forth in SEQ ID NO:360). Also, basic residues atpositions corresponding to positions 11 and 81 set forth in SEQ IDNO:360 also are found in other link modules as determined by alignment.

HA binding proteins containing link modules for use in the methodsprovided herein include, but are not limited to, TSG-6 (e.g. set forthin SEQ ID NO: 206 as the precursor and in SEQ ID NO:222 as the matureprotein lacking a signal sequence; or the LM set forth in SEQ ID NO:207,360, 417 or 418, which represent various lengths of the LM as reportedin the literature), stabilin-1 (e.g. set forth in SEQ ID NO:223 or themature form thereof; or the LM set forth in SEQ ID NO:371), stabilin-2(e.g. set forth in SEQ ID NO:224 or the mature form thereof; or the LMset forth in SEQ ID NO:372), CD44 (e.g. set forth in SEQ ID NO:227 orthe mature form thereof; or the LM set forth in SEQ ID NO:375), LYVE-1(e.g. set forth in SEQ ID NO:228 or the mature form thereof; or the linkmodule set forth in SEQ ID NO:376), HAPLN1 (e.g. HAPLN1-1 and HAPLN1-2;e.g., set forth in SEQ ID NO:229 or the mature form thereof; or the LMor LMs set forth in SEQ ID NO:377 or 378), HAPLN2 (e.g. HAPLN2-1 andHAPLN2-2; e.g. set forth in SEQ ID NO:230 or the mature form thereof; orthe LM or LMs set forth in SEQ ID NO: 379 or 380), HAPLN3 (e.g. HAPLN3-1and HAPLN3-2; e.g. set forth in SEQ ID NO:231 or the mature formthereof; or the LM or LMs set forth in SEQ ID NO:381 or 382), HAPLN4(e.g. HAPLN4-1 and HAPLN4-2; e.g. set forth in SEQ ID NO:232 or themature form thereof; or the LM or LMs set forth in SEQ ID NO:383 or384), aggrecan (e.g. aggrecan 1, aggrecan 2, aggrecan 3 and aggrecan 4;e.g. set forth in SEQ ID NO:233 or the mature form thereof; or the LM orLMs set forth in SEQ ID NO: 385, 386, 387 or 388), versican (e.g.versican 1 and versican 2; e.g. set forth in SEQ ID NO:235 or the matureform thereof; or the LM or LMs set forth in SEQ ID NO:391 or 392),brevican (e.g. brevican 1 and brevican 2; e.g. set forth in SEQ IDNO:234 or the mature form thereof; or the LM or LMs set forth in SEQ IDNO:389 or 390), neurocan (e.g. neurocan 1 and neurocan 2; e.g. set forthin SEQ ID NO:236 or the mature form thereof; e.g. the LM or LMs setforth in SEQ ID NO:393 or 394) and phosphacan (e.g. set forth in SEQ IDNO:340 or the mature form thereof). Exemplary of an HABP provided foruse in the methods herein is TSG-6.

In particular examples herein, the HABP used in the methods hereincontains at least one link module, and in some cases contains at leasttwo or at least three link modules. The HABP can be a full-length HABPcontaining a link module. For example, the companion diagnostic reagentfor use in the method herein can contain a sequence of amino acids setforth in any of SEQ ID NOS:206, and 223-236, the mature form thereof, ora sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%,84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to a sequence set forth in any of SEQ ID NOS: 206, and 223-236.For example, the HABP for use as a companion diagnostic herein can be afull-length TSG-6 having the sequence of amino acids set forth in SEQ IDNO:222, or a sequence of amino acids that exhibits at least 65%, 70%,75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to a sequence set forth in SEQ ID NO:222.

In other examples, the companion diagnostic reagent for use in themethods herein contains only the link module or sufficient portion of alink module to bind to HA derived from a full-length HABP set forth inany of SEQ ID NOS: 206, and 223-236 or the mature form thereof (lackingthe signal sequence). In some examples, the HABP containing a linkmodule or modules is not the complete sequence of an HABP set forth inany of SEQ ID NOS: 206, and 223-236 or the mature form thereof (lackingthe signal sequence). It is understood that the portion of an HABP orlink module is generally a contiguous sequence of amino acids that isgenerally at least 50 amino acids in length, 60, 70, 80, 90, 100, 200,300 or more amino acids. In some examples, the link module or modules isthe only HABP portion of the companion diagnostic binding molecule. Forexample, the companion diagnostic reagent for use in the method hereincontains only a portion of a full-length HABP and has a sequence ofamino acids set forth in any of SEQ ID NOS:207, 360, 361, 371-394 and416-418 or a sequence of amino acids that exhibits at least 65%, 70%,75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to a sequence set forth in any of SEQ ID NOS: 207,360, 361, 371-394 and 416-418.

In examples herein, the companion diagnostic reagent for use in themethods herein contains a G1 domain or sufficient portion thereof tobind to specifically bind to HA. The HABP containing the G1 domain canbe derived from a full-length HABP set forth in any of SEQ ID NOS:233-236 or the mature form thereof. In some examples, the HABPcontaining the G1 domain is not the complete sequence of an HABP setforth in any of SEQ ID NOS:233-236 or mature form thereof. It isunderstood that portion of an HABP containing a G1 domain is generally acontiguous sequence of amino acids that is generally at least 100 aminoacids in length, such as 150, 200, 250, 300, 400, or more amino acids.In some examples, the G1 domain is the only HABP portion of thecompanion diagnostic binding molecule. For example, the companiondiagnostic reagent for use in the method herein contains only a portionof a full-length HABP and has a G1 domain having a sequence of aminoacids set forth in any of SEQ ID NOS: 423-426 or a sequence of aminoacids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence setforth in any of SEQ ID NOS: 423-426.

In some examples, the companion diagnostic can contain more than onelink module, such as two or three link modules. The link modules can befrom the same or different HABP. The companion diagnostics can containlink modules that are linked directly or indirectly to form a singlepolypeptide. In other examples, the companion diagnostics can containlink modules that are set forth as separate polypeptides that arechemically linked, such as via a disulfide bond. Exemplary of an HABPfragment provided for use in the methods herein is the link domain ofTSG-6 (TSG-6-LM), or a portion thereof sufficient to bind to HA.

In some examples, the HABP is a multimer containing two or more linkmodules that are linked directly or indirectly via a multimerizationdomain to effect the formation of dimer or trimer molecules and thegeneration of multiple HA binding sites. For example, a companiondiagnostic for use in the methods herein is one that is generated byexpression of a nucleic acid molecule encoding the link module set forthin any one of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418 or asequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toa sequence set forth in any of SEQ ID NOS: 207, 360, 361, 371-394 and416-418 linked directly or indirectly to a nucleic acid encoding amultimerization domain, such as an Fc portion of an immunoglobulin.Hence, the resulting HABP multimer or LM-multimer contains a firstpolypeptide set forth in any one of SEQ ID NOS: 207, 360, 361, 371-394and 416-418 or a sequence of amino acids that exhibits at least 65%,70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to a sequence set forth in any of SEQ ID NOS: 207,360, 361, 371-394 and 416-418 linked directly or indirectly to amultimerization domain; and a second polypeptide set forth in any one ofSEQ ID NOS: 207, 360, 361, 371-394 and 416-418 or a sequence of aminoacids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence setforth in any of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418 linkeddirectly or indirectly to a multimerization domain. The sequence of thelink module in the first and second polypeptide can be the same ordifferent. Exemplary of an HABP multimer provided for use in the methodsherein is a multimer containing two polypeptide chains, whereby eachcontains the TSG-6-LM, variant thereof or sufficient portion thereof tobind HA linked directly or indirectly to a multimerization domain thateffects multimerization. For example, provided herein for use in themethods is a TSG-6-LM:Fc molecule (see e.g. SEQ ID NO:212 or 215).

A description of exemplary HABPs containing link domains, includingstructure and function description, is provided below. Any of thedescribed HABPs or portions thereof, such as a fragment containing onlya link domain or sufficient portion thereof to bind HA, can be used as acompanion diagnostic reagent in the methods herein. It is understoodthat reference to amino acids, including to a specific sequence setforth as a SEQ ID NO used to describe domain organization of a linkdomain or other domain are for illustrative purposes and are not meantto limit the scope of the embodiments provided. It is understood thatpolypeptides and the description of domains thereof are theoreticallyderived based on homology analysis and alignments with similarmolecules. Thus, the exact locus can vary, and is not necessarily thesame for each HABP. Hence, the specific domain, such as specific linkdomain, can be several amino acids (one, two, three or four) longer orshorter.

a. Type A: TSG-6 Sub-Group

Provided herein as a companion diagnostic for use in the methods hereinare HABPs that are members of the Type A sub-group that contain a singlelink module that binds to hyaluronan. Type A HABPs bind to HA with aminimum chain length of six sugars, hexasaccharide (HA₆), or greater.Members of the Type A sub-group that can be used as companiondiagnostics in the methods provided herein include, but are not limitedto, TSG-6, Stabilin-1, Stabilin-2, CAB61358 and KIAA0527, link modulesthereof, or sufficient portions of a link module that binds HA.

i. TSG-6

Exemplary of a Type A sub-group HABP provided for use as a companiondiagnostic reagent in the methods provided herein is TSG-6, or a linkmodule thereof, a sufficient portion of a link module to bind to HA,variants thereof or multimers thereof. Tumor necrosis factor-StimulatedGene-6 (TSG-6, tumor necrosis factor alpha-induced protein 6, TNFAIP6;SEQ ID NO:206) is a ˜35 kDa secreted glycoprotein composed of a singleN-terminal link module and C-terminal CUB domain. Expression of TSG-6 isinduced in many cell types by inflammatory mediators, includingcytokines and growths factors. Via its link module, TSG-6 is a potentinhibitor of polymorphonuclear leukocyte migration. TSG-6 forms a stablecomplex with the serine protease inhibitor Inter-alpha-Inhibitor (IαI)and potentiates the anti-plasmin activity of IαI. TSG-6 also isimportant for the formation and remodeling of HA-rich pericellular coatsand extracellular matrices.

The human TSG-6 transcript (SEQ ID NO:205) is normally translated toform a 277 amino acid precursor peptide (SEQ ID NO:206) containing a 17amino acid signal sequence at the N-terminus. The mature TSG-6 (setforth in SEQ ID NO:222), therefore, is a 260 amino acid proteincontaining amino acids 18-277 of SEQ ID NO:206 (Lee et al. (1992) J CellBiol 116:545-557). TSG-6 is composed of two main domains, the linkmodule and the CUB domain. The link module of TSG-6 is variouslyreported in the literature to be located at amino acids 35-129, 36-128,36-129 or 36-132 of SEQ ID NO:206 (set forth as SEQ ID NOS: 207, 360,417 or 418, respectively). It is understood that reference to loci of adomain can vary by several amino acids due to differences in alignments.Hence, for purposes herein, a TSG-6-LM is one set forth in any of SEQ IDNOS: 207, 360, 417 or 418 or that varies from such sequence by one, twoor three amino acids. The CUB domain is located at amino acids 135-246of SEQ ID NO:206. Human TSG-6 has two potential N-linked glycans atresidues N118 and N258 of SEQ ID NO:206. In addition, residues T259 andT262 of SEQ ID NO:206 are phosphorylated (Molina et al. (2007) Proc NatlAcad Sci USA 104:2199-2204). Human TSG-6 has eight native cysteineswhich form four disulfide bonds at residues C58-C127, C82-C103,C135-C161 and C188-C210 of preprotein TSG-6 (SEQ ID NO:206).

TSG-6 link module (SEQ ID NO:360) has a relatively small size and awell-characterized structure. The three dimensional structure of theTSG-6 link domain was determined and found to have the same fold asother known link modules, containing two alpha helices and twoantiparallel beta sheets arranged around a large hydrophobic core (Kohdaet al. (1996) Cell 86:767-775). In addition, the interaction of the linkmodule of TSG-6 and HA has been studied revealing that the aromaticrings of Tyr12, Tyr59, Phe70, Tyr78, Trp88 and basic residues Lys11,Lys72, Asp77, Arg 81, and Glu86 of the link domain of TSG-6 (SEQ IDNO:360) are important for binding to HA (see, e.g., Kahmann et al.(2000) Structure 8:763-774; Mahoney et al. (2001) J Biol Chem276:22764-22771; Kohda et al. (1996) Cell, 88:767-775; Blundell et al.(2003) J Biol Chem 278:49261-49270; Lesley et al. (2004) J Biol Chem279:25745-25754; Blundell et al. (2005) J Biol Chem 280:18189-18201).Structural studies also show that there is only a single HA-binding sitecontained in the link module, which is localized to one region of themolecule based on the structural map of residues Lys11, Tyr12, Tyr59,Phe70 and Tyr78 that are most directly implicated in HA binding (seee.g. Mahoney et al. (2001) J Biol Chem 276:22764-22771).

The link module of TSG-6 exhibits binding activity to severalglycosaminoglycans. For example, studies have revealed binding of thelink module to HA, chondroitin-4-sulphate (C4S), G1-domain of theproteoglycan aggrecan, heparin and the bikunin chain of IαI (see e.g.,Milner et al. (2003) Journal of Cell Science, 116:1863-1873; Mahoney etal. (2005) Journal of Biological Chemistry, 280:27044-27055). Thebinding of TSG-6 to heparin and HA is mediated by a distinct bindingsite in the LM of TSG-6. The residues involved in TSG-6-LM binding tohyaluronan are Lys11, Tyr12, Tyr59, Phe70 and Tyr78, whereby the mutantsK11Q, Y12F, Y59F, F70V and Y78F have between 10- and 100-fold lowerHA-binding affinity compared to wildtype; the residues in the TSG-6-LMinvolved in binding to heparin are Lys20, Lys34, Lys41, Lys54, Arg56 andArg84, whereby the mutants K20A, K34A, K41A and K54A exhibit impairedheparin binding properties; and the residues involved in TSG-6-LMbinding to bikunin is overlapping with but not identical to the HAbinding site (Mahoney et al. (2005) Journal of Biological Chemistry,280:27044-27055).

Binding of TSG-6 to hyaluronan is pH dependent, with binding activityexhibited at acidic pH of about or pH 5.6 to 6.4, such as or about pH5.8 to pH 6.0.

TSG-6 polypeptides, HA binding domains thereof, e.g., TSG-6 linkmodules, or fragments thereof sufficient to bind to HA provided hereinfor use as a companion diagnostic in the in the methods herein caninclude any of SEQ ID NOS: 206, 207, 222, 360, 417 or 418, or variantsthereof such as variants that exhibit at least 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to any one of SEQ ID NOS: 206, 207, 222, 360, 417 or 418.Exemplary variants include, for example, species variants, allelicvariants and variants that contain conservative and non-conservativeamino acid mutations. Natural allelic variants of human TSG-6 include,for example, TSG-6 containing the amino acid replacement Q144R (SEQ IDNO:407, Nentwich et al. (2002) J Biol Chem 277:15354-15362). TSG-6 ishighly conserved among species with mouse and human protein being >94%identical. Species variants of TSG-6 or HA binding fragments thereof foruse as a companion diagnostic in the methods provided herein alsoinclude, but are not limited to, mouse (SEQ ID NO:252), rabbit (SEQ IDNO:253), bovine (SEQ ID NO:254), horse (SEQ ID NO:409), chimpanzee (SEQID NO:408), dog (SEQ ID NO:410), mouse (SEQ ID NO:411), chicken (SEQ IDNO:412), frog Xenopus laevis (SEQ ID NO:413), zebra fish (SEQ IDNO:414), mature forms thereof or link modules or sufficient portionsthereof to bind HA.

Variants of TSG-6 or HA binding fragments thereof for use in theprovided methods include variants with an amino acid modification thatis an amino acid replacement (substitution), deletion or insertion.Exemplary modifications are amino acid replacements such as an aminoacid replacement at any of amino acid residues 4, 6, 8, 13, 20, 29, 34,41, 45, 54, 67, 72 or 96 corresponding to residues in the TSG-6 setforth in SEQ ID NO: 360, 417 or 418. The replacement amino acid can beany other amino acid residue. Exemplary amino acid replacements of TSG-6polypeptides or HA binding fragments thereof provided herein for use asa companion diagnostic reagent in the methods provided herein includemodified TSG-6 polypeptides or HA-binding fragments thereof that containat least one amino acid replacement corresponding to H4K, H4S, E6A, E6K,R8A, K13A, K20A, H29K, K34A, K41A, H45S, K54A, N67L, N67S, K72A, H96K,K34A/K54A or K20A/K34A/K41A corresponding to residues in the TSG-6 setforth in SEQ ID NO: 360, 417 or 418 (see, e.g., Mahoney et al. (2005) JBiol Chem 280:27044-27055, Blundell et al. (2007) J Biol Chem282:12976-12988, Lesley et al. (2004) J Biol Chem 279:25745-25754,Kahmann et al. (2000) Structure 8:763-774). It is understood thatresidues important or otherwise required for the binding of TSG-6 to HA,such as any described above or known to one of skill in the art, aregenerally invariant and cannot be changed. Thus, for example, amino acidresidues 11, 12, 59, 70, 78 and 81 of SEQ ID NO: 360 in the link moduleof TSG-6 are generally invariant and are not altered. Further, it isunderstood that amino acid modifications that result in improper foldingor perturbation of the folding of the link module are generallyinvariant. Thus, for example, a modified TSG-6 provided for use in themethods herein will not contain any one or more of the amino acidmodifications H4S, H29A, H45A, H45K, R56A, D77A, R84A and D89A of SEQ IDNO:360 (Mahoney et al. (2005) J Biol Chem 280:27044-27055, Blundell etal. (2007) J Biol Chem 282:12976-12988, Lesley et al. (2004) J Biol Chem279:25745-25754).

In particular, the modification, for example amino acid replacement orreplacements, is one that confers an altered, such as improved, activitycompared to a TSG-6 not containing the modification. Such variantsinclude those that contain amino acid modifications that enhance thebinding affinity of TSG-6 to HA, increase the specificity of TSG-6 forHA, and/or increase the solubility of TSG-6. For example provided hereinfor use in the methods herein are TSG-6 variants, HA binding domains, orportions thereof sufficient to bind to HA that increase the specificityof TSG-6 for HA by decreasing the binding of TSG-6 to otherglycosaminoglycans, including heparin, chondroitin-4-sulfate, heparansulfate and dermatan sulfate. Binding to the other glycosaminoglycanthat is not hyaluronan can be reduced at least 1.2-fold, 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 100-fold or more compared to bindingof TSG-6-LM not containing the modification. For example, providedherein is a mutant TSG-6-LM containing amino acid replacement(s) atamino acid residues 20, 34, 41, 54, 56, 72 and/or 84, and in particularat amino acid residues 20, 34, 41, and/or 54 (corresponding to aminoacid residues set forth in SEQ ID NO:206). The replacement amino acidcan be to any other amino acid residue, and generally is to a non-basicamino acid residue. For example, amino acid replacement can be to Asp(D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Ala (A), Val (V), Ile(I), Leu (L), Met (M), Phe (F), Tyr (Y) or Trp (W). The amino acidreplacement or replacements confer decreased binding to heparin. Forexample, variants that decrease the ability of TSG-6 to bind to heparinare known to one of skill in the art. Such variants are those thatinclude at least one mutation corresponding to K20A, K34A, K41A andK54A, including variants K34A/K54A or K20A/K34A/K41A (Mahoney et al.(2005) J Biol Chem 280:27044-27055). Exemplary variants that decrease orreduce binding to heparin are variant TSG-6-LM set forth in SEQ IDNO:361 or 416.

Exemplary of a TSG-6 polypeptide provided herein for use in the methodsprovided herein is a TSG-6 polypeptide that contains at least an HAbinding domain, for example, a TSG-6 link module. Thus, provided hereinis a TSG-6 link module, or variant thereof, for use in the providedmethods. Exemplary of such a polypeptide reagent is one that has asequence of amino acids set forth in SEQ ID NO: 207, 360, 361, 416, 417or 418, or has a sequence of amino acids that exhibits at least 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 95%, 99% ormore sequence identity to any of SEQ ID NOS: 207, 360, 361, 416, 417 or418. For example, the TSG-6 link module can be modified to alter itsspecificity, affinity or solubility, as long as it retains its abilityto bind to HA.

In yet another example, the affinity of the TSG-6 link module isincreased by dimerization or multimerization, such as, for example, byfusion to a multimerization domain, such as an Fc domain (see Section C3below). Hence, the TSG-6 link module can be modified to produce amultimer containing two or more link modules that are linked directly orindirectly via a multimerization domain to effect the formation of dimeror trimer molecules and the generation of multiple HA binding sites. Forexample, a companion diagnostic for use in the methods herein is onethat is generated by expression of a nucleic acid molecule encoding thelink module set forth in any one of SEQ ID NOS: 207, 360, 361, 417 or418 or a nucleic acid encoding a link module having a sequence of aminoacids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence setforth in any of SEQ ID NOS: 207, 360, 361, 417 or 418 linked directly orindirectly to a nucleic acid encoding a multimerization domain, such asan Fc portion of an immunoglobulin. Hence, the resulting TSG-6-LMmultimer contains a first polypeptide set forth in any one of SEQ IDNOS: 207, 360, 361, 417 or 418 or a sequence of amino acids thatexhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to a sequence set forth in anyof SEQ ID NOS: 207, 360, 361, 417 or 418 linked directly or indirectlyto a multimerization domain; and a second polypeptide set forth in anyone of SEQ ID NOS: 207, 360, 361, 417 or 418 or a sequence of aminoacids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence setforth in any of SEQ ID NOS: 207, 360, 361, 417 or 418 linked directly orindirectly to a multimerization domain. Generally, the LM or sufficientportion thereof to effect HA binding is the only TSG-6 portion of themultimer. For example, provided herein for use in the methods is aTSG-6-LM:Fc molecule (see e.g. SEQ ID NO:212 or 215).

In another example, the TSG-6 link module is linked to a Fc domain toincrease its solubility (see Section C3 below).

ii. Stabilin-1 and Stabilin-2

Exemplary of a Type A sub-group HABP provided for use as a companiondiagnostic reagent in the methods provided herein is Stabilin-1 orStabilin-2, or a link module thereof, a sufficient portion of a linkmodule to bind to HA, variants thereof or multimers thereof. Stabilin-1(also called STAB1, CLEVER-1, KIAA0246, FEEL-1, FEX-1 and FELE-1; SEQ IDNO:223) and Stabilin-2 (also called STAB2, FEEL-2, CD-44 like precursorFELL2, DKFZp434E0321, FEX2, and hyaluronan receptor forendocytosis/HARE; SEQ ID NO:224) are type I transmembrane members of afamily of fasciclin-like hyaluronan (HA) receptor homologs. Both containseven fasciclin-like adhesion domains, multiple EGF-like repeats, andhyaluronan-binding link modules. Both Stabilin-1 and Stabilin-2 areexpressed on sinusoidal endothelium and macrophages, though each isfunctionally distinct. Stabilin-1 is involved in two intracellulartrafficking pathways: receptor mediated endocytosis and recycling; andshuttling between the endosomal compartment and trans-Golgi network(TGN). Stabilin-2 acts as a scavenger receptor for HA and AGE-modifiedproteins.

The precursor sequence of Stabilin-1 is set forth in SEQ ID NO:223. Thelink module of Stabilin-1 is located at 2208-2300 of SEQ ID NO:223 andis set forth in SEQ ID NO:371. The precursor sequence of Stabilin-2 isset forth in SEQ ID NO:224 and the link module of Stabilin-2 is locatedat amino acids 2198-2290 of SEQ ID NO:224 and is set forth in SEQ IDNO:372.

Stabilin-1 or Stabilin-2 polypeptides, HA binding domains thereof, e.g.,Stabilin-LM modules or fragments thereof sufficient to bind to HAprovided herein for use as a companion diagnostic in the methods hereininclude the link module set forth in SEQ ID NO:371 or 372, or variantsthereof that exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any oneof SEQ ID NOS: 371 or 372. The variants include variants that exhibitspecific binding to HA. Variants include allelic variants, speciesvariants or other variants containing an amino acid modification (e.g.to increase affinity or specificity to HA). Species variants ofstabilin-1 provided for use in the methods herein include, but are notlimited to, mouse (SEQ ID NO:255) and bovine (SEQ ID NO:256) and speciesvariants of stabilin-2 provided for use in the methods herein include,but are not limited to, mouse (SEQ ID NO:257) and rat (SEQ ID NO:258).

Also provided herein for use as a companion diagnostic in the methodsherein is a Stablin-1-LM or Stabilin-1-LM multimer that exhibitsincreased affinity for HA. For example, a companion diagnostic for usein the methods herein is one that is generated by expression of anucleic acid molecule encoding the link module set forth in any one ofSEQ ID NOS: 371 or 372 or a nucleic acid encoding a link module having asequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toa sequence set forth in any of SEQ ID NOS: 371 or 372 linked directly orindirectly to a nucleic acid encoding a multimerization domain, such asan Fc portion of an immunoglobulin. Hence, the resulting LM multimercontains a first polypeptide set forth in any one of SEQ ID NOS: 371 or372 or a sequence of amino acids that exhibits at least 65%, 70%, 75%,80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to a sequence set forth in any of SEQ ID NOS: 371 or 372 linkeddirectly or indirectly to a multimerization domain; and a secondpolypeptide set forth in any one of SEQ ID NOS: 371 or 372 or a sequenceof amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to asequence set forth in any of SEQ ID NOS: 371 or 372 linked directly orindirectly to a multimerization domain.

b. Type B: CD44 Sub-Group

Provided herein as a companion diagnostic reagent for use in the methodsherein are HABPs that are members of the Type B sub-group having anHA-binding domain that contains a single link module with N- andC-terminal extensions that binds to hyaluronan. Unlike the HA bindingdomain of the Type A/TSG-6 sub-group, the flanking sequences of the linkdomain are essential for the structural integrity of the Type B domainand are required for binding to HA. Members of the Type B sub-group ofHABPs for use in the methods provided herein include, but are notlimited to, CD44 and LYVE-1, or HA binding fragments thereof.

i. CD44

A Type B sub-group HABP provided for use in the methods herein is CD44,HA binding domains of CD44 or portions thereof sufficient to bind to HA.CD44 is an 80- to 250-kDa Type I transmembrane glycoprotein that bindshyaluronan and a variety of extracellular and cell-surface ligands. CD44has diverse functions and is involved in attachment, organization andturnovers of the extracellular matrix and mediates the migration oflymphocytes during inflammation. The ability of CD44 to interact with HAis regulated by factors, including receptor clustering and changes inglycosylation of the extracellular domain. CD exists in numerousisoforms due to alternative splicing of 10 variant exons, all of whichcontain the hyaluronan binding domain containing the link module. Anexemplary CD44 full length sequence is set forth in SEQ ID NO:227. Thehyaluronan binding domain of CD44 is approximately 160 amino acids inlength (SEQ ID NO: 341) and contains the link module flanked by N- andC-terminal extensions linked by a disulfide bond (Cys9 and Cys110 of theCD44 HA binding domain set forth in SEQ ID NO: 341). Arg41 and Arg78 arecritical for HA binding (corresponding to amino acids Arg22 and Arg59 ofthe CD44 HA binding domain set forth in SEQ ID NO: 341) and Tyr42 andTyr79 (corresponding to amino acids Tyr23 and Tyr60 of the CD44 HAbinding domain set forth in SEQ ID NO: 341) are essential for CD44functional activity. The link domain of CD44 is set forth in SEQ IDNO:375. Thus provided herein for use in the methods herein are fragmentsof CD44 that retain the ability to bind to HA, for example, a fragmentof CD44 that contains a link domain and N- and C-terminal flankingdomains or a sufficient portion thereof to effect binding to HA.

Also provided herein for use in the provided methods are variants,including allelic variants, species variants and other variantscontaining an amino acid modification, as long as the variants retaintheir ability to bind to HA. Species variants of CD44 for use in themethods provided herein include, but are not limited to, mouse (SEQ IDNO:259), rat (SEQ ID NO:260), bovine (SEQ ID NO:261), dog (SEQ IDNO:262), horse (SEQ ID NO:263), hamster (SEQ ID NO:264), baboon (SEQ IDNO:265) and golden hamster (SEQ ID NO:266). Variants of CD44, or HAbinding fragments thereof, for use in the provided methods includevariants that have an amino acid modification and that exhibit analtered, such as improved, activity compared to a CD44 not containingthe modification. Such variants include those that contain amino acidmodifications that enhance the binding affinity of CD44 to HA, increasethe specificity of CD44 for HA, and/or increase the solubility of CD44.

ii. LYVE-1

Provided herein for use in the methods provided herein is a Type Bsub-group HABP that is LYVE-1, HA binding domains of LYVE-1 or portionsthereof sufficient to bind to HA. Lymphatic Vessel EndothelialHyaluronan (HA) Receptor-1 (LYVE-1, also called CRSBP-1, HAR, and XLKD1;SEQ ID NO:228) is a 60-kDa type I transmembrane glycoprotein that isexpressed on both the lumenal and ablumenal surfaces of lymphaticendothelium, and also on hepatic blood sinusoidal endothelia. LYVE-1participates in HA internalization for degradation and transport of HAfrom tissues into the lumen of lymphatic vessels. LYVE-1-directed HAlocalization to lymphatic surfaces also affects aspects of the immuneresponse or tumor metastases. The link module of LYVE-1 is located atamino acids 40-129 of SEQ ID NO:228 and is set forth in SEQ ID NO:376.Thus provided herein for use in the methods herein are fragments ofLYVE-1 that retain the ability to bind to HA, for example, a fragment ofLYVE-1 that contains a link domain and N- and C-terminal flankingdomains or a sufficient portion thereof to effect binding to HA.

Also provided herein for use in the provided methods are variants,including allelic variants, species variants and other variantscontaining an amino acid modification, as long as the variants retaintheir ability to bind to HA. Species variants of LYVE-1 include, but arenot limited to, mouse (SEQ ID NO:267) and bovine (SEQ ID NO:268).Variants of LYVE-1, or HA binding fragments thereof, for use in theprovided methods include variants that have an amino acid modificationand that exhibit an altered, such as improved, activity compared to aLYVE-1 not containing the modification. Such variants include those thatcontain amino acid modifications that enhance the binding affinity ofLYVE-1 to HA, increase the specificity of LYVE-1 for HA, and/or increasethe solubility of LYVE-1.

c. Type C: Link Protein Sub-Group

Provided herein for use as a companion diagnostic reagent in the methodsherein are HABPs that are members of the Type C sub-group having an HAbinding domain that contains an immunoglobulin (Ig) domain, whichmediates binding between link protein and other Type C HA bindingproteins, and two link modules, both of which are required for bindingto HA. The Ig domain and two link modules collectively make up the G1domain of Type C HABPs. Members of the Type C sub-group of HABPs for usein the methods provided herein include, but are not limited to,HAPLN1/link protein, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican,brevican, neurocan, and phosphacan, or HA binding fragments thereof.

i. HAPLN/Link Protein Family

The Hyaluronan and Proteoglycan Link Protein (HAPLN) family is made upof four secreted proteoglycans that bind hyaluronan and contain oneIg-type C2-set domain and two link domains.

(1) HAPLN1

A Type C sub-group HABP provided herein for use in the methods isHAPLN1, HA binding domains of HAPLN1 or portions thereof sufficient tobind to HA. Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1, alsocalled as link protein and CRTL1; SEQ ID NO: 229) contributes toextracellular matrix stability and flexibility by stabilizinginteractions of HA with chondroitin sulfate proteoglycans. HALPN1contains two link modules (amino acids 159-253 and amino acids 260-350of SEQ ID NO: 229) that bind to HA and an Ig module (amino acids 53-160of SEQ ID NO: 229) that binds to the Ig module of the G1 domain ofaggrecan. HAPLN1 stabilizes associations of HA with aggrecan by forminga ternary complex containing an HA linear backbone with perpendicularlyattached aggrecan and HAPLN1. Aggrecan and HAPLN1 lie parallel to eachother, while HA runs between the two HAPLN1 link modules and the twoaggrecan link modules. The complex creates a gel-like substance withresistance to deformation. HAPLN1 also stabilizes the interaction of HAwith other chondroitin sulfate proteoglycans, such as versican,neurocan, and brevican, which also have G1 domains containing an Igmodule and two link modules, similar to aggrecan.

The G1 domain of HAPLN1 contains the Ig domain and the 2 link modules.The Ig domain of the G1 domain of HAPLN1 is located at amino acids53-160 of SEQ ID NO:229. The link modules of the G1 domain of HAPLN1 arelocated at amino acids 159-253 and 259-350 of SEQ ID NO:229 and are setforth in SEQ ID NOS:377 and 378. Thus, provided herein for use in themethods herein are fragments of HAPLN1 that retain the ability to bindto HA, for example, a fragment of HAPLN1 that contains the G1 domain ora sufficient portion thereof to effect binding to HA. For example,provided herein for use in the methods herein is an HA binding fragmentof HAPLN1 that contains at least the two link modules.

Typically, for use as a diagnostic for the detection of HA, HAPLN1 isprovided in combination with another HA binding protein that containsthe HA-binding region, such as, for example, the G1 domain of anotherType C HABP, such as aggrecan, versican, brevican, neurocan, orphosphacan.

Also provided herein for use in the provided methods are variants,including allelic variants, species variants and other variantscontaining an amino acid modification, as long as the variants retaintheir ability to bind to HA. Species variants of HAPLN1 include, but arenot limited to, bovine (SEQ ID NO:269 and 273), mouse (SEQ ID NO:270),rat (SEQ ID NO:271), chicken (SEQ ID NO:272), horse (SEQ ID NO:274) andpig (SEQ ID NO:275). Variants of HAPLN1, or HA binding fragmentsthereof, for use in the provided methods include variants that have anamino acid modification and that exhibit an altered, such as improved,activity compared to an HAPLN1 not containing the modification. Suchvariants include those that contain amino acid modifications thatenhance the binding affinity of HAPLN1 to HA, increase the specificityof HAPLN1 for HA, and/or increase the solubility of HAPLN1.

(2) HAPLN2

Provided herein for use in the methods provided herein is a Type Csub-group HABP that is HAPLN2, HA binding domains of HAPLN2 or portionsthereof sufficient to bind to HA. Hyaluronan and Proteoglycan LinkProtein 2 (HAPLN2; SEQ ID NO: 230), also known as brain link protein 1,is predominantly expressed in brain. The G1 domain of HAPLN2 containsthe Ig domain and the 2 link modules. The Ig domain of the G1 domain ofHAPLN2 is located at amino acids 49-149 of SEQ ID NO:230. The linkmodules of the G1 domain of HAPLN2 are located at amino acids 148-241and 247-337 of SEQ ID NO:230 and are set forth in SEQ ID NOS:379 and380.

Thus, provided herein for use in the methods herein are fragments ofHAPLN2 that retain the ability to bind to HA, for example, a fragment ofHAPLN2 that contains the G1 domain or a sufficient portion thereof toeffect binding to HA. For example, provided herein for use in themethods herein is an HA binding fragment of HAPLN2 that contains atleast the two link modules. Typically, for use as a diagnostic for thedetection of HA, HAPLN2 is provided in combination with another HAbinding protein that contains the HA-binding region, such as, forexample, the G1 domain of another Type C HABP, such as aggrecan,versican, brevican, neurocan, or phosphacan.

Also provided herein for use in the provided methods are variants,including allelic variants, species variants and other variantscontaining an amino acid modification, as long as the variants retaintheir ability to bind to HA. Species variants of HAPLN2 include, but arenot limited to, mouse (SEQ ID NO:276), rat (SEQ ID NO:277) and bovine(SEQ ID NO:278). Variants of HAPLN2, or HA binding fragments thereof,for use in the provided methods include variants that have an amino acidmodification and that exhibit an altered, such as improved, activitycompared to an HAPLN2 not containing the modification. Such variantsinclude those that contain amino acid modifications that enhance thebinding affinity of HAPLN2 to HA, increase the specificity of HAPLN1 forHA, and/or increase the solubility of HAPLN2.

(3) HAPLN3

A Type C sub-group HABP provided herein for use in the methods herein isHAPLN3, HA binding domains of HAPLN3 or portions thereof sufficient tobind to HA. Hyaluronan and Proteoglycan Link Protein 3, (HAPLN3; SEQ IDNO:231), functions in hyaluronic acid binding and cell adhesion. HAPLN3is upregulated in breast cancer and, thus, may be related to cancerdevelopment and metastasis. The G1 domain of HAPLN3 contains the Igdomain and the 2 link modules. The Ig domain of the G1 domain of HAPLN3is located at amino acids 62-167 of SEQ ID NO:231. The link modules ofthe G1 domain of HAPLN3 are located at amino acids 166-260 and 266-357of SEQ ID NO:231 and are set forth in SEQ ID NOS:381 and 382.

Thus, provided herein for use in the methods herein are fragments ofHAPLN3 that retain the ability to bind to HA, for example, a fragment ofHAPLN3 that contains the G1 domain or a sufficient portion thereof toeffect binding to HA. For example, provided herein for use in themethods herein is an HA binding fragment of HAPLN3 that contains atleast the two link modules. Typically, for use as a diagnostic for thedetection of HA, HAPLN3 is provided in combination with another HAbinding protein that contains the HA-binding region, such as, forexample, the G1 domain of another Type C HABP, such as aggrecan,versican, brevican, neurocan, or phosphacan.

Also provided herein for use in the methods herein are variants,including allelic variants, species variants and other variantscontaining an amino acid modification, as long as the variants retaintheir ability to bind to HA. Species variants of HAPLN3 include, but arenot limited to, mouse (SEQ ID NO:279), rat (SEQ ID NO:280) and bovine(SEQ ID NO:281). Variants of HAPLN3, or HA binding fragments thereof,for use in the provided methods include variants that have an amino acidmodification and that exhibit an altered, such as improved, activitycompared to an HAPLN3 not containing the modification. Such variantsinclude those that contain amino acid modifications that enhance thebinding affinity of HAPLN3 to HA, increase the specificity of HAPLN3 forHA, and/or increase the solubility of HAPLN3.

(4) HAPLN4

Provided herein for use in the methods herein is a Type C sub-group HABPthat is HAPLN4, HA binding domains of HAPLN4 or portions thereofsufficient to bind to HA. Hyaluronan and Proteoglycan Link Protein 4,(HAPLN4; SEQ ID NO:232), also known as brain link protein 2, ispredominantly expressed in brain. HAPLN4 participates in the developmentof the perineuronal matrix. Human and mouse HAPLN4 share 91% amino acidsequence identity. The G1 domain of HAPLN4 contains the Ig domain andthe 2 link modules. The Ig domain of the G1 domain of HAPLN4 is locatedat amino acids 60-164 of SEQ ID NO:232. The link modules of the G1domain of HAPLN4 are located at amino acids 163-267 and 273-364 of SEQID NO:232 and are set forth in SEQ ID NOS:383 and 384.

Thus, provided herein for use in the methods herein are fragments ofHAPLN4 that retain the ability to bind to HA, for example, a fragment ofHAPLN4 that contains the G1 domain or a sufficient portion thereof toeffect binding to HA. For example, provided herein for use in themethods herein is an HA binding fragment of HAPLN4 that contains atleast the two link modules. Typically, for use as a diagnostic for thedetection of HA, HAPLN4 is provided in combination with another HAbinding protein that contains the HA-binding region, such as, forexample, the G1 domain of another Type C HABP, such as aggrecan,versican, brevican, neurocan, or phosphacan.

Also provided herein for use in the provided methods are variants ofHAPLN4, including allelic variants, species variants and other variantscontaining an amino acid modification, as long as the variants retaintheir ability to bind to HA. Species variants of HAPLN4 include, but arenot limited to, mouse (SEQ ID NO:282), bovine (SEQ ID NO:283) and rat(SEQ ID NO:284). Variants of HAPLN4, or HA binding fragments thereof,for use in the provided methods include variants that have an amino acidmodification and that exhibit an altered, such as improved, activitycompared to an HAPLN4 not containing the modification. Such variantsinclude those that contain amino acid modifications that enhance thebinding affinity of HAPLN4 to HA, increase the specificity of HAPLN4 forHA, and/or increase the solubility of HAPLN4.

(5) Aggrecan

Provided herein for use in the methods herein is a Type C sub-group HABPthat is aggrecan, HA binding domains of aggrecan or portions thereofsufficient to bind to HA. Aggrecan (SEQ ID NO:233) belongs to thechondroitin sulfate (CS) proteoglycan family, which also includesversican, brevican, neurocan, and phosphacan. Each aggrecan moleculecontains approximately 100 and 30 keratan sulfate and glycosaminoglycan(GAG) side chains, respectively. Aggrecan non-covalently associates withhyaluronan via the link modules and an Ig domain in its N-terminus. Itis the most abundant proteoglycan in cartilage, and contributes to theload-bearing capacity of this tissue.

The G1 domain of aggrecan is located at amino acids 45-352 of SEQ IDNO:233. The Ig domain of the G1 domain of aggrecan is located at aminoacids 45-154 of SEQ ID NO:233 and is set forth in SEQ ID NO:423. Thelink modules of the G1 domain of aggrecan are located at amino acids153-247 and 254-349 of SEQ ID NO:233 and are set forth in SEQ ID NOS:385and 386. Link modules 3 and 4 are set forth in SEQ ID NOS:387 and 388.Thus, provided herein for use in the methods herein are fragments ofaggrecan that retain the ability to bind to HA, for example, a fragmentof aggrecan that contains the G1 domain or a sufficient portion thereofto effect binding to HA.

Also provided herein for use in the provided methods are variants ofaggrecan, including allelic variants, species variants and othervariants containing an amino acid modification, as long as the variantsretain their ability to bind to HA. Species variants of aggrecaninclude, but are not limited to, pig (SEQ ID NO:285), chicken (SEQ IDNO:286), mouse (SEQ ID NO:287), bovine (SEQ ID NO:288), dog (SEQ IDNO:289), rat (SEQ ID NO:290) and rabbit (SEQ ID NO:291). Variants ofaggrecan, or HA binding fragments thereof, for use in the providedmethods include variants that have an amino acid modification and thatexhibit an altered, such as improved, activity compared to an aggrecannot containing the modification. Such variants include those thatcontain amino acid modifications that enhance the binding affinity ofaggrecan to HA, increase the specificity of aggrecan for HA, and/orincrease the solubility of aggrecan.

(6) Brevican

Provided herein for use in the methods herein is a Type C sub-group HABPthat is brevican, HA binding domains of brevican or portions thereofsufficient to bind to HA. Brevican (SEQ ID NO:234) is a 160 kDa memberof the aggrecan/versican proteoglycan family of matrix proteins. It isbrain-derived and serves as a linker between hyaluronan and other matrixmolecules such as the tenascins and fibulins. The G1 domain of brevicanis located at amino acids 51-356 of SEQ ID NO:234 and is set forth inSEQ ID NO:424. The Ig domain of the G1 domain of brevican is located atamino acids 51-158 of SEQ ID NO:234. The link modules of the G1 domainof brevican are located at amino acids 157-251 and 258-353 of SEQ IDNO:234 and are set forth in SEQ ID NOS:389 and 390. Thus, providedherein for use in the methods herein are fragments of brevican thatretain the ability to bind to HA, for example, a fragment of brevicanthat contains the G1 domain or a sufficient portion thereof to effectbinding to HA. For example, provided herein for use in the methodsherein is an HA binding fragment of brevican that contains at least thetwo link modules.

Also provided herein for use in the provided methods are variants ofbrevican, including allelic variants, species variants and othervariants containing an amino acid modification, as long as the variantsretain their ability to bind to HA. Species variants of brevicaninclude, but are not limited to, rat (SEQ ID NO:292), mouse (SEQ IDNO:293), bovine (SEQ ID NO:294) and cat (SEQ ID NO:295). Variants ofbrevican, or HA binding fragments thereof, for use in the providedmethods include variants that have an amino acid modification and thatexhibit an altered, such as improved, activity compared to a brevicannot containing the modification. Such variants include those thatcontain amino acid modifications that enhance the binding affinity ofbrevican to HA, increase the specificity of brevican for HA, and/orincrease the solubility of brevican.

(7) Versican

Provided herein for use in the methods herein is a Type C sub-group HABPthat is versican, HA binding domains of versican or portions thereofsufficient to bind to HA. Versican (SEQ ID NO:235) is a largeextracellular matrix proteoglycan that is present in a variety oftissues. It plays important structural roles, forming loose, hydratedmatrices during development and disease. It also interacts directly orindirectly with cells to regulate such physiological processes as celladhesion, survival, proliferation, and motility. The G1 domain ofversican is located at amino acids 38-349 of SEQ ID NO:235 and is setforth in SEQ ID NO:425. The Ig domain of the G1 domain of versican islocated at amino acids 38-151 of SEQ ID NO:235. The link modules of theG1 domain of versican are located at amino acids 150-244 and 251-346 ofSEQ ID NO:235 and are set forth in SEQ ID NOS:391 and 392. Thus,provided herein for use in the methods herein are fragments of versicanthat retain the ability to bind to HA, for example, a fragment ofversican that contains the G1 domain or a sufficient portion thereof toeffect binding to HA. For example, provided herein for use in themethods herein is an HA binding fragment of versican that contains atleast the two link modules.

Also provided herein for use in the provided methods are variants ofversican, including allelic variants, species variants and othervariants containing an amino acid modification, as long as the variantsretain their ability to bind to HA. Species variants of versicaninclude, but are not limited to, mouse (SEQ ID NO:296), rat (SEQ IDNO:297), pig-tailed macaque (SEQ ID NO:298), bovine (SEQ ID NO:299) andchicken (SEQ ID NO:300). Variants of versican, or HA binding fragmentsthereof, for use in the provided methods include variants that have anamino acid modification and that exhibit an altered, such as improved,activity compared to a versican not containing the modification. Suchvariants include those that contain amino acid modifications thatenhance the binding affinity of versican to HA, increase the specificityof versican for HA, and/or increase the solubility of versican.

(8) Neurocan

Provided herein for use in the methods herein is a Type C sub-group HABPthat is neurocan, HA binding domains of neurocan or portions thereofsufficient to bind to HA. Neurocan, also known as CSPG3 and 1D1 (SEQ IDNO:236), is a secreted chondroitin sulfate proteoglycan that isprimarily expressed in the central nervous system. Human Neurocan ispredicted to be cleaved following Met635, resulting in N-terminal(Neurocan-130) and C-terminal (Neurocan-C) fragments. Neurocan andNeurocan-C are produced by astrocytes and accumulate in the matrixsurrounding axonal bundles and neuronal cell bodies. Neurocan-130 isfound mainly in the glial cell cytoplasm. Neurocan inhibits neuronaladhesion and neurite outgrowth through interactions with a variety ofmatrix and transmembrane molecules. The G1 domain of neurocan is locatedat amino acids 53-359 of SEQ ID NO:236 and is set forth in SEQ IDNO:426. The Ig domain of the G1 domain of neurocan is located at aminoacids 53-161 of SEQ ID NO:236. The link modules of the G1 domain ofneurocan are located at amino acids 160-254 and 261-356 of SEQ ID NO:236and are set forth in SEQ ID NOS:393 and 394. Thus, provided herein foruse in the methods herein are fragments of neurocan that retain theability to bind to HA, for example, a fragment of neurocan that containsthe G1 domain or a sufficient portion thereof to effect binding to HA.For example, provided herein for use in the methods herein is an HAbinding fragment of neurocan that contains at least the two linkmodules.

Also provided herein for use in the provided methods are variants,including allelic variants, species variants and other variantscontaining an amino acid modification, as long as the variants retaintheir ability to bind to HA. Species variants of neurocan include, butare not limited to, mouse (SEQ ID NO:301), rat (SEQ ID NO:302) andchimpanzee (SEQ ID NO:303). Variants of neurocan, or HA bindingfragments thereof, for use in the provided methods include variants thathave an amino acid modification and that exhibit an altered, such asimproved, activity compared to a neurocan not containing themodification. Such variants include those that contain amino acidmodifications that enhance the binding affinity of neurocan to HA,increase the specificity of neurocan for HA, and/or increase thesolubility of neurocan.

(9) Phosphacan

Provided herein for use in the method provided herein is a Type Csub-group HABP that is phosphacan, HA binding domains of phosphacan orportions thereof sufficient to bind to HA. Phosphacan (SEQ ID NO:340) achondroitin sulfate proteoglycan isolated from rat brain that binds toneurons and neural cell-adhesion molecules and modulate cellinteractions and other developmental processes in nervous tissue throughheterophilic binding to cell-surface and extracellular matrix molecules,and by competition with ligands of the transmembrane phosphatase.Phosphacan has 76% identity to the extracellular portion of a humanreceptor-type protein tyrosine phosphatase (RPTP zeta/beta) andrepresent an mRNA splicing variant of the larger transmembrane protein.

Also provided herein for use in the methods herein are variants,including allelic variants, species variants and other variantscontaining an amino acid modification, as long as the variants retaintheir ability to bind to HA. Species variants of phosphacan include, butare not limited to rat phosphacan (SEQ ID NO:237). Variants ofphosphacan, or HA binding fragments thereof, for use in the providedmethods include variants that have an amino acid modification and thatexhibit an altered, such as improved, activity compared to a phosphacannot containing the modification. Such variants include those thatcontain amino acid modifications that enhance the binding affinity ofphosphacan to HA, increase the specificity of phosphacan for HA, and/orincrease the solubility of phosphacan.

2. HA Binding Proteins without Link Modules

In some examples, provided herein for use in the methods herein are HAbinding proteins that do not contain link modules. HA binding proteinswithout link modules for use in the methods provided herein include, butare not limited to, HABP1/C1QBP, layilin, RHAMM, IαI, CDC37, PHBP,SPACR, SPACRCAN, CD38, IHABP4 and PEP-1, or HA binding fragmentsthereof.

a. HABP1/C1QBP

Provided herein for use in the methods herein is a hyaluronan bindingprotein 1, HA binding domains of HABP1 or portions thereof sufficient tobind to HA. Hyaluronan binding protein 1 (HABP1; SEQ ID NO:240), alsoknown as C1qBP/C1qR and p32, is a ubiquitous acidic glycoprotein thatfunctions in spermatogenesis and as a receptor for proinflammatorymolecules. HABP1 binds extracellular hyaluronan, vitronectin, complementcomponent C1q, HMW kininogen, and bacterial and viral proteins.Intracellular HABP1 binds to molecules containing the C1q globulardomain, multiple isoforms of PKC, mitochondrial Hrk, adrenergic andGABA-A receptors, the mRNA splicing factor ASF/SF2, and the CBFtranscription factor.

Also provided herein for use in the methods herein are variants,including allelic variants, species variants and other variantscontaining an amino acid modification, as long as the variants retaintheir ability to bind to HA. Variants of HABP1, or HA binding fragmentsthereof, for use in the provided methods include variants that have anamino acid modification and that exhibit an altered, such as improved,activity compared to an HABP1 not containing the modification. Suchvariants include those that contain amino acid modifications thatenhance the binding affinity of HABP1 to HA, increase the specificity ofHABP1 for HA, and/or increase the solubility of HABP 1.

b. Layilin

Provided herein for use in the methods herein is a layilin, HA bindingdomains of layilin or portions thereof sufficient to bind to HA. Layilin(SEQ ID NOS:238 and 239) is transmembrane protein with homology toC-type lectins and is named after the L-A-Y-I-L-I six amino acid motifin its transmembrane segment. Layilin binds specifically to hyaluronanand is found in the extracellular matrix of most animal tissues and inbody fluids. It thus can modulate cell behavior and functions duringtissue remodeling, development, homeostasis, and diseases.

Also provided herein for use in the methods herein are variants,including allelic variants, species variants and other variantscontaining an amino acid modification, as long as the variants retaintheir ability to bind to HA. Species variants of layilin include, butare not limited to, mouse (SEQ ID NO:304), Chinese hamster (SEQ IDNO:305) and rat (SEQ ID NO:306). Variants of layilin, or HA bindingfragments thereof, for use in the provided methods include variants thathave an amino acid modification and that exhibit an altered, such asimproved, activity compared to a layilin not containing themodification. Such variants include those that contain amino acidmodifications that enhance the binding affinity of layilin to HA,increase the specificity of layilin for HA, and/or increase thesolubility of layilin.

c. RHAMM

Provided herein for use in the methods herein is a RHAMM, HA bindingdomains of RHAMM or portions thereof sufficient to bind to HA. Thereceptor for HA-mediated motility (RHAMM; SEQ ID NO:242) is amembrane-associated protein, ranging is size from ˜59 to 80 kDa. RHAMMis expressed on most cell types and functions to mediate adhesion andcell motility in response to HA binding. Also provided herein for use inthe methods herein are variants, including allelic variants, speciesvariants and other variants containing an amino acid modification, aslong as the variants retain their ability to bind to HA. Speciesvariants of RHAMM include, but are not limited to, mouse (SEQ ID NO:307)and rat (SEQ ID NO:308). Variants of RHAMM, or HA binding fragmentsthereof, for use in the provided methods include variants that have anamino acid modification and that exhibit an altered, such as improved,activity compared to a RHAMM not containing the modification. Suchvariants include those that contain amino acid modifications thatenhance the binding affinity of RHAMM to HA, increase the specificity ofRHAMM for HA, and/or increase the solubility of HABP1.

d. Others

Other HABPs that bind to HA some of which contain hyaluronan bindingdomains that can be used in the methods provided herein include, but arenot limited to, IαI (SEQ ID NOS:243-245), CDC37 (SEQ ID NO:250), PHBP(SEQ ID NO:251), SPACR (SEQ ID NO:246), SPACERCAN (SEQ ID NO:247), CD38(SEQ ID NO:248), IHABP4 (SEQ ID NO:249) and PEP-1 (SEQ ID NO:241), or HAbinding domains or portions thereof sufficient to bind to HA. Alsoprovided herein for use in the methods herein are variants, includingallelic variants, species variants and other variants containing anamino acid modification, as long as the variants retain their ability tobind to HA. Species variants include, but are not limited to, IαI frommouse (SEQ ID NOS:309-311) and bovine (SEQ ID NOS:312-314), CDC37 fromBaker's yeast (SEQ ID NO:326), fruit fly (SEQ ID NO:327), rat (SEQ IDNO:328), mouse (SEQ ID NO:329), fission yeast (SEQ ID NO:330), fruit fly(SEQ ID NO:331), chicken (SEQ ID NO:332), bovine (SEQ ID NO:333),Candida albicans (SEQ ID NO:334). C. elegans (SEQ ID NO:335) and greenpufferfish (SEQ ID NO:336), SPACR from chicken (SEQ ID NO:315) and mouse(SEQ ID NO:316), SPACRCAN from mouse (SEQ ID NO:317), rat (SEQ IDNO:318) and chicken (SEQ ID NO:319), CD38 from mouse (SEQ ID NO:320),rat (SEQ ID NO:321) and rabbit (SEQ ID NO:322), IHABP4 from mouse (SEQID NO:324) and chicken (SEQ ID NO:325), and PHBP from mouse (SEQ IDNO:337), rat (SEQ ID NO:338) and bovine (SEQ ID NO:339). Variants ofHABPs, or HA binding fragments thereof, for use in the provided methodsinclude variants that have an amino acid modification and that exhibitan altered, such as improved, activity compared to an HABP notcontaining the modification. Such variants include those that containamino acid modifications that enhance the binding affinity of an HABP toHA, increase the specificity of an HABP for HA, and/or increase thesolubility of an HABP, such as an IαI, CDC37, PHBP, SPACR, SPACRCAN,CD38, IHABP4 and PEP-1, or HA binding fragments thereof.

3. Modifications of HA Binding Proteins

Modified HABPs are provided herein to improve one or more properties ofHABPs for use in the methods provided herein. Such properties includemodifications increase protein expression in mammalian expressionsystems, improve biophysical properties such as stability andsolubility, improve protein purification and detection and/or increaseaffinity to HA via dimerization of the fusion protein.

a. Multimers of HABP

HABPs provided for use in the methods herein can be linked directly orindirectly to a multimerization domain. The presence of amultimerization domain can generate multimers of HABPs or HA bindingdomains thereof to increase HA binding sites on a molecule. This canresult in increased affinity of the HABP for HA. For example, affinityof an HABP multimer can be increased 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more compared to an HABPpolypeptide not containing a multimerization domain. Affinity of an HABPmultimer for HA, when represented as the dissociation constant (Kd), isgenerally at least less than or less than or 1×10⁻⁸ M to 1×10⁻¹⁰ M, suchas at least less than or less than or 9×10⁻⁹ M, 8×10⁻⁹ M, 7×10⁻⁹ M,6×10⁻⁹ M, 5×10⁻⁹ M, 4×10⁻⁹ M, 3×10⁻⁹ M, 2×10⁻⁹ M, 1×10⁻⁹ M, 9×10⁻¹⁰ M,8×10⁻¹⁰ M, 7×10⁻¹⁰ M, 6×10⁻¹⁰ M, 5×10⁻¹⁰ M, 4×10⁻¹⁰ M, 3×10⁻¹⁰ M,2×10⁻¹⁰ M, 1×10⁻¹⁰ M or lower Kd.

Provided herein are multimers that include an HA binding domain orsufficient portion thereof to bind HA of a first HABP and an HA bindingdomain or sufficient portion thereof to bind HA of a second HABP, wherethe first and second HA-binding domain are linked directly or indirectlyvia a linker to a multimerization domain. The first and secondHA-binding domain can be from the same HABP or from a different HABP.For example, if the HA-binding domain is the same, then homodimers orhomotrimers can be generated. If the HA binding domain is different,then heterodimers or heterotrimers can be generated. For example, HAbinding domains, such as a link domain or module, of HABPs can becovalently-linked, non-covalently-linked or chemically linked to formmultimers of two or more HA binding domains. The link modules can belinked to form dimers, trimers, or higher multimers. In some instances,multimers can be formed by dimerization of two or more HABP polypeptidesthat each contain an HA binding domain.

Any portion of an HABP including an HA binding domain can be used as amultimer partner. For example, any of the HABPs described above, orthose set forth in any of SEQ ID NOS:206-207, 222-340, 407-414 or anyportion of an HABP, including an HA binding domain, for example, a linkdomain or module and variants thereof, including any HA binding domainsset forth in any of SEQ ID NOS: 341 and 371-394 can be used to generatechimeric HABP polypeptides, wherein all or part of the HABP polypeptideis linked to a multimerization domain. Typically, at least one, butsometimes both, of the HABP portions is all or a portion of an HABPsufficient to bind HA linked to a multimerization domain. Examples ofHABPs, or portions thereof, for use as multimerization partners aredescribed herein above and are set forth in any of SEQ ID NOS: 206-207,222-341, 371-394, 407-414, 416-418 or 423-426. In some examples, atleast one of the multimer partners is all or part of the HABP includingthe HA binding domain. For example, exemplary of multimeric HABPpolypeptides is a multimer formed between the HA binding domain (e.g.link domain or link module), or portion thereof, of aggrecan, versican,neurocan, brevican, phosphacan, HAPLN1, HAPLN2, HAPLN3, HAPLN4,stabilin-1, stabilin-2, CAB61358, KIAA0527 or TSG-6 protein.Additionally, a chimeric HABP polypeptide for use in the formation of anHABP multimer can include hybrid HABP polypeptides linked to amultimerization domain. Exemplary of a multimer provided herein is amultimer, such as a homodimer, generated by multimerization of the linkmodule (LM) of TSG-6 or sufficient portion thereof that binds to HA.

Multimerization between two HABP polypeptides can be spontaneous, or canoccur due to forced linkage of two or more polypeptides. In one example,multimers can be linked by disulfide bonds formed between cysteineresidues on different HABP polypeptides or domain or sufficient portionsthereof that bind to HA. In another example, multimers can include anHABP polypeptide or domain or sufficient portion thereof to bind to HAjoined via covalent or non-covalent interactions to peptide moietiesfused to the each polypeptide. Such peptides can be peptide linkers(e.g. spacers) or peptides that have the property of promotingmultimerization. In an additional example, multimers can be formedbetween two polypeptides through chemical linkage, such as for example,by using heterobifunctional linkers.

i. Peptide Linkers

Peptide linkers can be used to produce HABP polypeptide multimers, suchas for example a multimer where at least one multimerization partnercontains an HA binding domain (e.g., a link domain or module). In oneexample, peptide linkers can be fused to the C-terminal end of a firstpolypeptide and the N-terminal end of a second polypeptide. Thisstructure can be repeated multiple times such that at least one,preferably 2, 3, 4, or more polypeptides are linked to one another viapeptide linkers at their respective termini. For example, a multimerpolypeptide can have a sequence Z₁-X-Z₂, where Z₁ and Z₂ are each asequence of all or part of an HABP including an HA binding domain andwhere X is a sequence of a peptide linker. In some instances, Z₁ and/orZ₂ is all of an HABP including an HA binding domain. In other instances,Z₁ and/or Z₂ is part of an HABP including an HA binding domain. Z₁ andZ₂ are the same or they are different. In another example, thepolypeptide has a sequence of Z₁-X-Z₂(-X-Z)_(n), where “n” is anyinteger, i.e. generally 1 or 2.

Typically, the peptide linker is of a sufficient length to allow one orboth HA binding domains to bind to a hyaluronan substrate or to permitinteraction between the HA binding domains (e.g. interaction of two Igmodules of the G1 HA binding domains of Type C HABPs). Examples ofpeptide linkers include, but are not limited to: -Gly-Gly-, GGGGG (SEQID NO:342), GGGGS or (GGGGS)n (SEQ ID NO:343), SSSSG or (SSSSG)n (SEQ IDNO:344), GKSSGSGSESKS (SEQ ID NO:345), GGSTSGSGKSSEGKG (SEQ ID NO: 346),GSTSGSGKSSSEGSGSTKG (SEQ ID NO: 347), GSTSGSGKPGSGEGSTKG (SEQ ID NO:348), EGKSSGSGSESKEF (SEQ ID NO: 349), or AlaAlaProAla or(AlaAlaProAla)n (SEQ ID NO:350), where n is 1 to 6, such as 1, 2, 3, or4. Exemplary linkers include:

(1) Gly4Ser with NcoI ends  (SEQ ID NO: 351)CCATGGGCGG CGGCGGCTCT GCCATGG (2) (Gly4Ser)2 with NcoI ends (SEQ ID NO: 352) CCATGGGCGG CGGCGGCTCT GGCGGCGGCG GCTCTGCCAT GG(3) (Ser4Gly)4 with NcoI ends  (SEQ ID NO: 353)CCATGGCCTC GTCGTCGTCG GGCTCGTCGT CGTCGGGCTCGTCGTCGTCG GGCTCGTCGT CGTCGGGCGC CATGG (4) (Ser4Gly)2 with NcoI ends (SEQ ID NO: 354) CCATGGCCTC GTCGTCGTCG GGCTCGTCGT CGTCGGGCGC CATGG

Linking moieties are described, for example, in Huston et al. (1988)PNAS 85:5879-5883, Whitlow et al. (1993) Protein Engineering 6:989-995,and Newton et al., (1996) Biochemistry 35:545-553. Other suitablepeptide linkers include any of those described in U.S. Pat. No.4,751,180 or 4,935,233, which are hereby incorporated by reference. Apolynucleotide encoding a desired peptide linker can be insertedbetween, and in the same reading frame as a polynucleotide encoding allor part of an HABP including an HA binding domain, using any suitableconventional technique. In one example, a fusion polypeptide has fromtwo to four HABP polypeptides, including one that is all or part of anHABP polypeptide including an HA binding domain, separated by peptidelinkers.

ii. Heterobifunctional Linking Agents

Linkage of an HABP polypeptide to another HABP polypeptide to create aheteromultimeric fusion polypeptide can be direct or indirect. Forexample, linkage of two or more HABP polypeptides can be achieved bychemical linkage or facilitated by heterobifunctional linkers, such asany known in the art or provided herein.

Numerous heterobifunctional cross-linking reagents that are used to formcovalent bonds between amino groups and thiol groups and to introducethiol groups into proteins, are known to those of skill in this art(see, e.g., the PIERCE CATALOG, ImmunoTechnology Catalog & Handbook,1992-1993, which describes the preparation of and use of such reagentsand provides a commercial source for such reagents; see, also, e.g.,Cumber et al. (1992) Bioconjugate Chem. 3:397-401; Thorpe et al. (1987)Cancer Res. 47:5924-5931; Gordon et al. (1987) Proc. Natl. Acad Sci.84:308-312; Walden et al. (1986) J. Mol. Cell Immunol. 2:191-197;Carlsson et al. (1978) Biochem. J. 173: 723-737; Mahan et al. (1987)Anal. Biochem. 162:163-170; Wawrzynczak et al. (1992) Br. J. Cancer66:361-366; Fattom et al. (1992) Infection & Immun. 60:584-589). Thesereagents can be used to form covalent bonds between the N-terminalportion of an HABP polypeptide including an HA binding domain andC-terminus portion of another HABP polypeptide including an HA bindingdomain or between each of those portions and a linker. These reagentsinclude, but are not limited to:N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP; disulfide linker);sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate(sulfo-LC-SPDP); succinimidyloxycarbonyl-α-methyl benzyl thiosulfate(SMBT, hindered disulfate linker); succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP); sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC);succinimidyl 3-(2-pyridyldithio)butyrate (SPDB; hindered disulfide bondlinker); sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate(SAED); sulfo-succinimidyl 7-azido-4-methylcoumarin-3-acetate (SAMCA);sulfosuccinimidyl-6-[alpha-methyl-alpha-(2-pyridyldithio)toluamido]-hexanoate(sulfo-LC-SMPT); 1,4-di-[3′-(2′-pyridyldithio)propionamido]butane(DPDPB); 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridylthio)toluene(SMPT, hindered disulfate linker);sulfosuccinimidyl-6-[α-methyl-α-(2-pyrimiyldi-thio)toluamido]hexanoate(sulfo-LC-SMPT); m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS);m-maleimidobenzoyl-N-hydroxysulfo-succinimide ester (sulfo-MBS);N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB; thioether linker);sulfosuccinimidyl-(4-iodoacetyl)amino benzoate (sulfo-SIAB);succinimidyl-4-(p-maleimi-dophenyl)butyrate (SMPB);sulfosuccinimidyl-4-(p-maleimido-phenyl)buty-rate (sulfo-SMPB);azidobenzoyl hydrazide (ABH). These linkers, for example, can be used incombination with peptide linkers, such as those that increaseflexibility or solubility or that provide for or eliminate sterichindrance. Any other linkers known to those of skill in the art forlinking a polypeptide molecule to another molecule can be employed.General properties are such that the resulting molecule binds to HA. Forin vivo diagnostic use of the HABP reagent, generally the linker must bebiocompatible for administration to animals, including humans.

iii. Polypeptide Multimerization Domains

Interaction of two or more HABP polypeptides can be facilitated by theirlinkage, either directly or indirectly, to any moiety or otherpolypeptide that are themselves able to interact to form a stablestructure. For example, separate encoded HABP polypeptide chains can bejoined by multimerization, whereby multimerization of the polypeptidesis mediated by a multimerization domain. Typically, the multimerizationdomain provides for the formation of a stable protein-proteininteraction between a first HABP polypeptide and a second HABPpolypeptide. HABP polypeptides include, for example, linkage (directlyor indirectly) of a nucleic acid encoding an HA binding domain (e.g. alink domain or module) of an HABP with a nucleic acid encoding amultimerization domain. Typically, at least one multimerization partneris a nucleic acid encoding all of part of an HABP including an HAbinding domain linked directly or indirectly to a multimerizationdomain, thus forming a chimeric molecule. Homo- or heteromultimericpolypeptides can be generated from co-expression of separate HABPpolypeptides. The first and second HABP polypeptides can be the same ordifferent.

Generally, a multimerization domain includes any capable of forming astable protein-protein interaction. The multimerization domains caninteract via an immunoglobulin sequence (e.g. Fc domain; see e.g.,International Patent Pub. Nos. WO 93/10151 and WO 2005/063816 US; U.S.Pub. No. 2006/0024298; U.S. Pat. No. 5,457,035), leucine zipper (e.g.from nuclear transforming proteins fos and jun or the proto-oncogenec-myc or from General Control of Nitrogen (GCN4)), a hydrophobic region,a hydrophilic region, or a free thiol which forms an intermoleculardisulfide bond between the chimeric molecules of a homo- orheteromultimer. In addition, a multimerization domain can include anamino acid sequence comprising a protuberance complementary to an aminoacid sequence comprising a hole, such as is described, for example, inU.S. Pat. No. 5,731,168; International Patent Pub. Nos. WO 98/50431 andWO 2005/063816; Ridgway et al. (1996) Protein Engineering, 9:617-621.Such a multimerization region can be engineered such that stericinteractions not only promote stable interaction, but further promotethe formation of heterodimers over homodimers from a mixture of chimericmonomers. Generally, protuberances are constructed by replacing smallamino acid side chains from the interface of the first polypeptide withlarger side chains (e.g., tyrosine or tryptophan). Compensatory cavitiesof identical or similar size to the protuberances are optionally createdon the interface of the second polypeptide by replacing large amino acidside chains with smaller ones (e.g., alanine or threonine). Exemplarymultimerization domains are described below.

An HABP polypeptide, such as for example any provided herein, includingany HA binding domain (e.g., a link domain or module) of an HABP, can bejoined anywhere, but typically via its N- or C-terminus, to the N- orC-terminus of a multimerization domain to form a chimeric polypeptideThe linkage can be direct or indirect via a linker. Also, the chimericpolypeptide can be a fusion protein or can be formed by chemicallinkage, such as through covalent or non-covalent interactions. Forexample, when preparing a chimeric polypeptide containing amultimerization domain, nucleic acid encoding all or part of an HABPincluding an HA binding domain can be operably linked to nucleic acidencoding the multimerization domain sequence, directly or indirectly oroptionally via a linker domain. Typically, the construct encodes achimeric protein where the C-terminus of the HABP polypeptide is joinedto the N-terminus of the multimerization domain. In some instances, aconstruct can encode a chimeric protein where the N-terminus of the HABPpolypeptide is joined to the N- or C-terminus of the multimerizationdomain.

A polypeptide multimer contains two chimeric proteins created bylinking, directly or indirectly, two of the same or different HABPpolypeptides directly or indirectly to a multimerization domain. In someexamples, where the multimerization domain is a polypeptide, a genefusion encoding the HABP-multimerization domain chimeric polypeptide isinserted into an appropriate expression vector. The resultingHABP-multimerization domain chimeric proteins can be expressed in hostcells transformed with the recombinant expression vector, and allowed toassemble into multimers, where the multimerization domains interact toform multivalent polypeptides. Chemical linkage of multimerizationdomains to HABP polypeptides can be effected using heterobifunctionallinkers as discussed above.

The resulting chimeric polypeptides, and multimers formed therefrom, canbe purified by any suitable method such as, for example, by affinitychromatography over Protein A or Protein G columns. Where two nucleicacid molecules encoding different HABP chimeric polypeptides aretransformed into cells, formation of homo- and heterodimers will occur.Conditions for expression can be adjusted so that heterodimer formationis favored over homodimer formation.

(1) Immunoglobulin Domain

Multimerization domains include those comprising a free thiol moietycapable of reacting to form an intermolecular disulfide bond with amultimerization domain of an additional amino acid sequence. Forexample, a multimerization domain can include a portion of animmunoglobulin molecule, such as from IgG1, IgG2, IgG3, IgG4, IgA, IgD,IgM, or IgE. Generally, such a portion is an immunoglobulin constantregion (Fc). Preparations of fusion proteins containing polypeptidesfused to various portions of antibody-derived polypeptides (includingthe Fc domain) has been described, see e.g., Ashkenazi et al. (1991)PNAS 88: 10535; Byrn et al. (1990) Nature, 344:667; and Hollenbaugh andAruffo, (2002)“Construction of Immunoglobulin Fusion Proteins,” inCurrent Protocols in Immunology, Ch. 10, pp. 10.19.1-10.19.11.

Antibodies bind to specific antigens and contain two identical heavychains and two identical light chains covalently linked by disulfidebonds. Both the heavy and light chains contain variable regions, whichbind the antigen, and constant (C) regions. In each chain, one domain(V) has a variable amino acid sequence depending on the antibodyspecificity of the molecule. The other domain (C) has a rather constantsequence common among molecules of the same class. The domains arenumbered in sequence from the amino-terminal end. For example, the IgGlight chain is composed of two immunoglobulin domains linked from N- toC-terminus in the order V_(L)-C_(L), referring to the light chainvariable domain and the light chain constant domain, respectively. TheIgG heavy chain is composed of four immunoglobulin domains linked fromthe N- to C-terminus in the order V_(H)-C_(H)1-C_(H)2-C_(H)3, referringto the variable heavy domain, contain heavy domain 1, constant heavydomain 2, and constant heavy domain 3. The resulting antibody moleculeis a four chain molecule where each heavy chain is linked to a lightchain by a disulfide bond, and the two heavy chains are linked to eachother by disulfide bonds. Linkage of the heavy chains is mediated by aflexible region of the heavy chain, known as the hinge region. Fragmentsof antibody molecules can be generated, such as for example, byenzymatic cleavage. For example, upon protease cleavage by papain, adimer of the heavy chain constant regions, the Fc domain, is cleavedfrom the two Fab regions (i.e. the portions containing the variableregions).

In humans, there are five antibody isotypes classified based on theirheavy chains denoted as delta (δ), gamma (γ), mu (μ), and alpha (α) andepsilon (ε), giving rise to the IgD, IgG, IgM, IgA, and IgE classes ofantibodies, respectively. The IgA and IgG classes contain the subclassesIgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Sequence differences betweenimmunoglobulin heavy chains cause the various isotypes to differ in, forexample, the number of C domains, the presence of a hinge region, andthe number and location of interchain disulfide bonds. For example, IgMand IgE heavy chains contain an extra C domain (C4), that replaces thehinge region. The Fc regions of IgG, IgD, and IgA pair with each otherthrough their Cγ3, Cδ3, and Cα3 domains, whereas the Fc regions of IgMand IgE dimerize through their Cμ4 and Cε4 domains. IgM and IgA formmultimeric structures with ten and four antigen-binding sites,respectively.

HABP immunoglobulin chimeric polypeptides provided herein include afull-length immunoglobulin polypeptide. Alternatively, theimmunoglobulin polypeptide is less than full length, i.e. containing aheavy chain, light chain, Fab, Fab₂, Fv, or Fc. In one example, the HABPimmunoglobulin chimeric polypeptides are assembled as monomers orhetero- or homo-multimers, and particularly as dimers or tetramers.Chains or basic units of varying structures can be utilized to assemblethe monomers and hetero- and homo-multimers. For example, an HABPpolypeptide can be fused to all or part of an immunoglobulin molecule,including all or part of C_(H), C_(L), V_(H), or V_(L) domain of animmunoglobulin molecule (see. e.g., U.S. Pat. No. 5,116,964). ChimericHABP polypeptides can be readily produced and secreted by mammaliancells transformed with the appropriate nucleic acid molecule. Thesecreted forms include those where the HABP polypeptide is present inheavy chain dimers; light chain monomers or dimers; and heavy and lightchain heterotetramers where the HABP polypeptide is fused to one or morelight or heavy chains, including heterotetramers where up to andincluding all four variable region analogues are substituted. In someexamples, one or more than one nucleic acid fusion molecule can betransformed into host cells to produce a multimer where the HABPportions of the multimer are the same or different. In some examples, anon-HABP polypeptide light-heavy chain variable-like domain is present,thereby producing a heterobifunctional antibody. In some examples, achimeric polypeptide can be made fused to part of an immunoglobulinmolecule lacking hinge disulfides, in which non-covalent or covalentinteractions of the two HABP polypeptide portions associate the moleculeinto a homo- or heterodimer.

(a) Fc Domain

Typically, the immunoglobulin portion of an HABP chimeric proteinincludes the heavy chain of an immunoglobulin polypeptide, most usuallythe constant domains of the heavy chain. Exemplary sequences of heavychain constant regions for human IgG sub-types are set forth in SEQ IDNOS: 355 (IgG1), SEQ ID NO: 356 (IgG2), SEQ ID NO: 357 (IgG3), and SEQID NO: 358 (IgG4). For example, for the exemplary heavy chain constantregion set forth in SEQ ID NO: 355, the C_(H)1 domain corresponds toamino acids 1-98, the hinge region corresponds to amino acids 99-110,the C_(H)2 domain corresponds to amino acids 111-223, and the C_(H)3domain corresponds to amino acids 224-330.

In one example, an immunoglobulin polypeptide chimeric protein caninclude the Fc region of an immunoglobulin polypeptide. Typically, sucha fusion retains at least a functionally active hinge, C_(H)2 and C_(H)3domains of the constant region of an immunoglobulin heavy chain. Forexample, a full-length Fc sequence of IgG1 includes amino acids 99-330of the sequence set forth in SEQ ID NO:355. An exemplary Fc sequence forhIgG1 is set forth in SEQ ID NO: 359, and contains almost all of thehinge sequence, and the complete sequence for the C_(H)2 and C_(H)3domain as set forth in SEQ ID NO:355. Another exemplary Fc polypeptideis the Fc polypeptide set forth in SEQ ID NO: 204. Another exemplary Fcpolypeptide is set forth in PCT application WO 93/10151, and is a singlechain polypeptide extending from the N-terminal hinge region to thenative C-terminus of the Fc region of a human IgG1 antibody (SEQ IDNO:359). The precise site at which the linkage is made is not critical:particular sites are well known and can be selected in order to optimizethe biological activity, secretion, or binding characteristics of theHABP polypeptide. For example, other exemplary Fc polypeptide sequencesbegin at amino acid C109 or P113 of the sequence set forth in SEQ ID NO:355 (see e.g., U.S. Pub. No. 2006/0024298).

In addition to hIgG1 Fc, other Fc regions also can be included in theHABP chimeric polypeptides provided herein. For example, where effectorfunctions mediated by Fc/FcγR interactions are to be minimized, fusionwith IgG isotypes that poorly recruit complement or effector cells, suchas for example, the Fc of IgG2 or IgG4, is contemplated. Additionally,the Fc fusions can contain immunoglobulin sequences that aresubstantially encoded by immunoglobulin genes belonging to any of theantibody classes, including, but not limited to IgG (including humansubclasses IgG1, IgG2, IgG3, or IgG4), IgA (including human subclassesIgA1 and IgA2), IgD, IgE, and IgM classes of antibodies. Further,linkers can be used to covalently link Fc to another polypeptide togenerate a Fc chimera.

Modified Fc domains also are contemplated herein for use in chimeraswith HABP polypeptides. In some examples, the Fc region is modified suchthat it exhibits altered binding to an FcR so has to result altered(i.e. more or less) effector function than the effector function of anFc region of a wild-type immunoglobulin heavy chain. Thus, a modified Fcdomain can have altered affinity, including but not limited to,increased or low or no affinity for the Fc receptor. For example, thedifferent IgG subclasses have different affinities for the FcγRs, withIgG1 and IgG3 typically binding substantially better to the receptorsthan IgG2 and IgG4. In addition, different FcγRs mediate differenteffector functions. FcγR1, FcγRIIa/c, and FcγRIIIa are positiveregulators of immune complex triggered activation, characterized byhaving an intracellular domain that has an immunoreceptor tyrosine-basedactivation motif (ITAM). FcγRIIb, however, has an immunoreceptortyrosine-based inhibition motif (ITIM) and is therefore inhibitory. Insome instances, an HABP polypeptide Fc chimeric protein provided hereincan be modified to enhance binding to the complement protein C1q.Further, an Fc can be modified to alter its binding to FcRn, therebyimproving the pharmacokinetics of an HABP-Fc chimeric polypeptide. Thus,altering the affinity of an Fc region for a receptor can modulate theeffector functions and/or pharmacokinetic properties associated by theFc domain. Modified Fc domains are known to one of skill in the art anddescribed in the literature, see e.g. U.S. Pat. No. 5,457,035; U.S.Patent Publication No. US 2006/0024298; and International PatentPublication No. WO 2005/063816 for exemplary modifications.

Typically, a polypeptide multimer is a dimer of two chimeric proteinscreated by linking, directly or indirectly, two of the same or differentHABP polypeptides to an Fc polypeptide. In some examples, a gene fusionencoding the HABP-Fc chimeric protein is inserted into an appropriateexpression vector. The resulting HABP-Fc chimeric proteins can beexpressed in host cells transformed with the recombinant expressionvector, and allowed to assemble much like antibody molecules, whereinterchain disulfide bonds form between the Fc moieties to yielddivalent HABP polypeptides.

The resulting chimeric polypeptides containing Fc moieties, andmultimers formed therefrom, can be easily purified by affinitychromatography over Protein A or Protein G columns. For the generationof heterodimers, additional steps for purification can be necessary. Forexample, where two nucleic acids encoding different HABP chimericpolypeptides are transformed into cells, the formation of heterodimersmust be biochemically achieved since HABP chimeric molecules carryingthe Fc-domain will be expressed as disulfide-linked homodimers as well.Thus, homodimers can be reduced under conditions that favor thedisruption of inter-chain disulfides, but do no effect intra-chaindisulfides. Typically, chimeric monomers with different HA-bindingdomain portions are mixed in equimolar amounts and oxidized to form amixture of homo- and heterodimers. The components of this mixture areseparated by chromatographic techniques. Alternatively, the formation ofthis type of heterodimer can be biased by genetically engineering andexpressing HABP fusion molecules that contain an HABP polypeptide,followed by the Fc-domain of hIgG, followed by either c-jun or the c-fosleucine zippers (see below). Since the leucine zippers formpredominantly heterodimers, they can be used to drive the formation ofthe heterodimers when desired.

HABP chimeric polypeptides containing Fc regions also can be engineeredto include a tag with metal chelates or other epitope. The tagged domaincan be used for rapid purification by metal-chelate chromatography,and/or by antibodies, to allow for detection of western blots,immunoprecipitation, or activity depletion/blocking in bioassays.

Exemplary HABP-Fc chimeric polypeptides include fusion protein of theTSG-6 link module (TSG-6-LM) and Fc. An exemplary TSG-6-LM-Fc is setforth in SEQ ID NO: 212, and encoded by a sequence of nucleotides setforth in SEQ ID NO: 211. In addition, HABP-Fc molecules, including forexample the exemplary TSG-6-Fc molecules, can optionally contain anepitope tag or a signal for expression and secretion. For example, theexemplary TSG-6-LM-Fc chimeric polypeptide set forth as SEQ ID NO:212contains human immunoglobulin light chain kappa (κ) leader signalpeptide sequence (SEQ ID NO: 210), an Fc fragment of the human IgG1heavy chain (SEQ ID NO:204) and a human TSG-6 link module (SEQ IDNO:207). The cDNA sequence encoding the TSG-6-LM-Fc chimeric polypeptideis set forth in SEQ ID NO: 211. The DNA encoding human IgG1 heavy chainand human TSG-6 link module regions are connected with a 6 bp AgeIrestriction enzyme cleavage site and a 12 bp sequence, GACAAAACTCAC (SEQID NO: 208), encoding four additional amino acids (DKTH; SEQ ID NO: 209)

(2) Leucine Zipper

Another method of preparing HABP polypeptide multimers for use in themethods provided herein involves use of a leucine zipper domain. Leucinezippers are peptides that promote multimerization of the proteins inwhich they are found. Typically, leucine zipper is a term used to referto a repetitive heptad motif containing four to five leucine residuespresent as a conserved domain in several proteins. Leucine zippers foldas short, parallel coiled coils, and are believed to be responsible foroligomerization of the proteins of which they form a domain. The dimerformed by a leucine zipper domain is stabilized by the heptad repeat,designated (abcdefg)n (see e.g., McLachlan and Stewart (1978) J. Mol.Biol. 98:293), in which residues a and d are generally hydrophobicresidues, with d being a leucine, which lines up on the same face of ahelix. Oppositely-charged residues commonly occur at positions g and e.Thus, in a parallel coiled coil formed from two helical leucine zipperdomains, the “knobs” formed by the hydrophobic side chains of the firsthelix are packed into the “holes” formed between the side chains of thesecond helix.

Exemplary leucine zippers for use as multimerization domains herein arederived from either of two nuclear transforming proteins, fos and jun,that exhibit leucine zipper domains, or the product of the murineproto-oncogene, c-myc. The leucine zipper domain is necessary forbiological activity (DNA binding) in these proteins. The products of thenuclear oncogenes fos and jun contain leucine zipper domains thatpreferentially form a heterodimer (O'Shea et al. (1989) Science,245:646; Turner and Tijian (1989) Science, 243:1689). For example, theleucine zipper domains of the human transcription factors c-jun andc-fos have been shown to form stable heterodimers with a 1:1stoichiometry (see e.g., Busch and Sassone-Corsi (1990) Trends Genetics,6:36-40; Gentz et al., (1989) Science, 243:1695-1699). Although jun-junhomodimers also have been shown to form, they are about 1000-fold lessstable than jun-fos heterodimers.

Thus, typically an HABP polypeptide multimer provided herein isgenerated using a jun-fos combination. Generally, the leucine zipperdomain of either c-jun or c-fos is fused in frame at the C-terminus ofan HABP of a polypeptide by genetically engineering fusion genes.Exemplary amino acid sequences of c-jun and c-fos leucine zippers areset forth in SEQ ID NOS:362 and 363, respectively. In some instances, asequence of a leucine zipper can be modified, such as by the addition ofa cysteine residue to allow formation of disulfide bonds, or theaddition of a tyrosine residue at the C-terminus to facilitatemeasurement of peptide concentration. Such exemplary sequences ofencoded amino acids of a modified c-jun and c-fos leucine zipper are setforth in SEQ ID NOS: 362 and 363, respectively. In addition, the linkageof an HABP polypeptide with a leucine zipper can be direct or can employa flexible linker domain, such as for example a hinge region of IgG, orother polypeptide linkers of small amino acids such as glycine, serine,threonine, or alanine at various lengths and combinations. In someinstances, separation of a leucine zipper from the C-terminus of anencoded polypeptide can be effected by fusion with a sequence encoding aprotease cleavage site, such as for example, a thrombin cleavage site.Additionally, the chimeric proteins can be tagged, such as for example,by a 6×His tag, to allow rapid purification by metal chelatechromatography and/or by epitopes to which antibodies are available,such as for example a myc tag, to allow for detection on western blots,immunoprecipitation, or activity depletion/blocking bioassays.

Another exemplary leucine zipper domain for use as a multimerizationdomain is derived from a nuclear protein that functions as atranscriptional activator of a family of genes involved in the GeneralControl of Nitrogen (GCN4) metabolism in S. cerevisiae. The protein isable to dimerize and bind promoter sequences containing the recognitionsequence for GCN4, thereby activating transcription in times of nitrogendeprivation. An exemplary sequence of a GCN4 leucine zipper capable offorming a dimeric complex is set forth in SEQ ID NO: 364. Amino acidsubstitutions in the a and d residues of a synthetic peptiderepresenting the GCN4 leucine zipper domain (i.e. amino acidsubstitutions in the sequence set forth as SEQ ID NO:364) have beenfound to change the oligomerization properties of the leucine zipperdomain. For example, when all residues at position a are changed toisoleucine, the leucine zipper still forms a parallel dimer. When, inaddition to this change, all leucine residues at position d also arechanged to isoleucine, the resultant peptide spontaneously forms atrimeric parallel coiled coil in solution. An exemplary sequence of sucha GNC4 leucine zipper domain capable of forming a trimer is set forth inSEQ ID NO:365. Substituting all amino acids at position d withisoleucine and at position a with leucine results in a peptide thattetramerizes. Such an exemplary sequence of a leucine zipper domain ofGCN4 capable of forming tetramers is set forth in SEQ ID NO:366.Peptides containing these substitutions are still referred to as leucinezipper domains since the mechanism of oligomer formation is believed tobe the same as that for traditional leucine zipper domains such as theGCN4 described above and set forth in SEQ ID NO:364.

(3) Protein-Protein Interaction Between Subunits

Exemplary of another type of multimerization domain for use in modifyingan HABP provided for use in the methods herein is one wheremultimerization is facilitated by protein-protein interactions betweendifferent subunit polypeptides. Exemplary of such a multimerizationdomain is derived from the mechanism of cAMP-dependent protein kinase(PKA) with its anchoring domain (AD) of A kinase anchor proteins (AKAP).Thus, a heteromultimeric HABP polypeptide can be generated by linking(directly or indirectly) a nucleic acid encoding an HABP polypeptide,such as an HA-binding domain of an HABP polypeptide, with a nucleic acidencoding an R subunit sequence of PKA (i.e. SEQ ID NO:367). This resultsin a homodimeric molecule, due to the spontaneous formation of a dimereffected by the R subunit. In tandem, another HABP polypeptide fusioncan be generated by linking a nucleic acid encoding another HABPpolypeptide to a nucleic acid sequence encoding an AD sequence of AKAP(i.e. SEQ ID NO:368). Upon co-expression of the two components, such asfollowing co-transfection of the HABP chimeric components in host cells,the dimeric R subunit provides a docking site for binding to the ADsequence, resulting in a heteromultimeric molecule. This binding eventcan be further stabilized by covalent linkages, such as for example,disulfide bonds. In some examples, a flexible linker residue can befused between the nucleic acid encoding the HABP polypeptide and themultimerization domain. In another example, fusion of a nucleic acidencoding an HABP polypeptide can be to a nucleic acid encoding an Rsubunit containing a cysteine residue incorporated adjacent to theamino-terminal end of the R subunit to facilitate covalent linkage (seee.g., SEQ ID NO:369). Similarly, fusion of a nucleic acid encoding apartner HABP polypeptide can be to a nucleic acid encoding an AD subunitalso containing incorporation of cysteine residues to both the amino-and carboxyl-terminal ends of AD (see e.g., SEQ ID NO:370).

iv. Other Multimerization Domains

Other multimerization domains that can be used to multimerize an HABPprovided for use in the methods herein are known to those of skill inthe art and are any that facilitate the protein-protein interaction oftwo or more polypeptides that are separately generated and expressed asHABP fusions. Examples of other multimerization domains that can be usedto provide protein-protein interactions between two chimericpolypeptides include, but are not limited to, the barnase-barstar module(see e.g., Deyev et al., (2003) Nat. Biotechnol. 21:1486-1492); use ofparticular protein domains (see e.g., Terskikh et al., (1997) Proc NatlAcad Sci USA 94: 1663-1668 and Muller et al., (1998) FEBS Lett.422:259-264); use of particular peptide motifs (see e.g., de Kruif etal., (1996) J. Biol. Chem. 271:7630-7634 and Muller et al., (1998) FEBSLett. 432: 45-49); and the use of disulfide bridges for enhancedstability (de Kruif et al., (1996) J. Biol. Chem. 271:7630-7634 andSchmiedl et al., (2000) Protein Eng. 13:725-734).

b. Mutations to Improve HA Binding

In a further example, provided herein for use in the methods herein areHABPs that are modified, such as by amino acid replacement, to exhibitincreased specificity for hyaluronan compared to other GAGs. Forexample, provided herein is a mutant TSG-6-LM containing amino acidreplacement(s) at amino acid residue 20, 34, 41, 54, 56, 72 and/or 84,and in particular at amino acid residue 20, 34, 41, and/or 54(corresponding to amino acid residues set forth in SEQ ID NO:360). Thereplacement amino acid can be to any other amino acid residue, andgenerally is to a non-basic amino acid residue. For example, amino acidreplacement can be to Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln(Q), Ala (A), Val (V), Ile (I), Leu (L), Met (M), Phe (F), Tyr (Y) orTrp (W). The amino acid replacement or replacements confer decreasedbinding to heparin. Binding can be reduced at least 1.2-fold, 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 100-fold or more compared to bindingof TSG-6-LM to heparin not containing the amino acid replacement.Exemplary of a TSG-6-LM mutant for use as a reagent in the methodprovided herein is K20A/K34A/K41A. Hence, for example, binding toheparin is reduced such that specificity to hyaluronan is increased. Themutant TSG-6-LM can be conjugated directly or indirectly to amultimerization domain to generate multimers. For example, exemplary ofa reagent for use in the methods herein is TSG-6-LM(K20A/K34A/K41A)-Fc.

c. Modifications of HA Binding Proteins for Detection

For use in the diagnostic methods provided herein, the HA bindingproteins can be modified to contain a detectable protein or a moiety tofacilitate detection.

i. Conjugation to Detectable Proteins or Moieties

The HA binding proteins for use in the diagnostic methods providedherein can be modified by conjugation to detectable moieties, including,but not limited to, peptides tags, radiolabels, fluorescent molecules,chemiluminescent molecules, bioluminescent molecules, Fc domains,biotin, enzymes that catalyze a detectable reaction or catalyzeformation of a detectable product and proteins that bind a detectablecompound. Detectable moieties, including proteins and compounds, ormoieties that facilitate detection are known to one of skill in the art.The detectable moieties can be used to facilitate detection and/orpurification of the HABP.

In one example, the HA binding protein is modified by conjugation to adetectable protein or to a protein that induces a detectable signal. Thedetectable protein or protein that induces a detectable signal can beselected from among a luciferase, a fluorescent protein, abioluminescent protein, a receptor or transporter protein that binds toand/or transports a contrast agent, chromophore, compound or ligand thatcan be detected. For example, the detectable protein or protein thatinduces a detectable signal is a green fluorescent protein (GFP) or ared fluorescent protein (RFP).

Detectable labels can be used in any of the diagnostic methods providedherein. Exemplary detectable labels include, for example,chemiluminescent moieties, bioluminescent moieties, fluorescentmoieties, radionuclides, and metals. Methods for detecting labels arewell known in the art. Such a label can be detected, for example, byvisual inspection, by fluorescence spectroscopy, by reflectancemeasurement, by flow cytometry, by X-rays, by a variety of magneticresonance methods such as magnetic resonance imaging (MRI) and magneticresonance spectroscopy (MRS). Methods of detection also include any of avariety of tomographic methods including computed tomography (CT),computed axial tomography (CAT), electron beam computed tomography(EBCT), high resolution computed tomography (HRCT), hypocycloidaltomography, positron emission tomography (PET), single-photon emissioncomputed tomography (SPECT), spiral computed tomography, and ultrasonictomography.

Exemplary of such proteins are enzymes that can catalyze a detectablereaction or catalyze formation of a detectable product, such as, forexample, luciferases, such as a click beetle luciferase, a Renillaluciferase, a firefly luciferase or beta-glucuronidase (GusA). Alsoexemplary of such proteins are proteins that emit a detectable signal,including fluorescent proteins, such as a green fluorescent protein(GFP) or a red fluorescent protein (RFP). A variety of DNA sequencesencoding proteins that can emit a detectable signal or that can catalyzea detectable reaction, such as luminescent or fluorescent proteins, areknown and can be used in the methods provided herein. Exemplary genesencoding light-emitting proteins include, for example, genes frombacterial luciferase from Vibrio harveyi (Belas et al., (1982) Science218:791-793), bacterial luciferase from Vibrio fischerii (Foran andBrown, (1988) Nucleic acids Res. 16:777), firefly luciferase (de Wet etal., (1987) Mol. Cell. Biol. 7:725-737), aequorin from Aequorea victoria(Prasher et al., (1987) Biochem. 26:1326-1332), Renilla luciferase fromRenilla renformis (Lorenz et al, (1991) Proc Natl Acad Sci USA88:4438-4442) and green fluorescent protein from Aequorea victoria(Prasher et al., (1987) Gene 111:229-233). The luxA and luxB genes ofbacterial luciferase can be fused to produce the fusion gene (Fab₂),which can be expressed to produce a fully functional luciferase protein(Escher et al., (1989) PNAS 86: 6528-6532).

Exemplary detectable proteins that can be conjugated to the HA bindingproteins for use in the diagnostic methods provided herein also includeproteins that can bind a contrasting agent, chromophore, or a compoundor ligand that can be detected, such as a transferrin receptor or aferritin; and reporter proteins, such as E. coli β-galactosidase,β-glucuronidase, xanthine-guanine phosphoribosyltransferase (gpt), andalkaline phosphatase. Also exemplary of detectable proteins are proteinsthat can specifically bind a detectable compound, including, but notlimited to receptors, metal binding proteins (e.g., siderophores,ferritins, transferrin receptors), ligand binding proteins, andantibodies.

The HABP also can be conjugated to a protein or peptide tag. In oneexample, the HA binding protein is conjugated to an Fc domain. Proteinand peptide tags also include, but are not limited to, HexaHis tag (SEQID NO:54), hemagglutinin tag (SEQ ID NO:420), FLAG tag (SEQ ID NO:55),c-myc tag (SEQ ID NO:419), VSV-G tag (SEQ ID NO:421), HSV tag (SEQ IDNO:422) and V5 tag (SEQ ID NO:415), chitin binding protein (CBP),maltose binding protein (MBP), and glutathione s-transferase (GST).

Detectable labels can be coupled or conjugated to an HABP throughrecombinant methods or by chemical methods. For example, conjugation canbe effected by linked the protein, directly or indirectly to a linkersuch as a peptide linker or a chemical linker. Linkers can bepolypeptide sequences, such as poly-Glycine sequences of between about 5and 200 amino acids. Proline residues can be incorporated into apolypeptide linker to prevent the formation of significant secondarystructural elements, i.e., α-helix/β-sheet, by the linker. An example ofa flexible linker is a polypeptide that includes a glycine chain with anintermediate proline. In other examples, a chemical linker is used toconnect synthetically or recombinantly produced binding and labelingdomain subsequences. Such flexible linkers are known to persons of skillin the art. For example, poly(ethylene glycol) linkers are availablefrom Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionallyhave amide linkages, sulfhydryl linkages, or heterofunctional linkages.

4. Selection of HA Binding Proteins for Diagnostic Use

An HA binding protein suitable for use as a diagnostic agent can beselected based on one or more desired properties or activities,including, but not limited to, specificity or affinity for HA,solubility, peptide stability, homogeneity, ease of expression andpurification, minimum batch to batch variations in the expressedpeptide, and low sample variability in HA binding and detection. In someexamples, a single polypeptide diagnostic agent is contemplated over adiagnostic with multiple polypeptide components. For example, a linkmodule that binds to HA in the absence of a complete link protein. Theability of an HABP provided herein to bind to hyaluronan can be assessedby methods well known in the art including, but not limited toELISA-based assays, competitive binding assays with HA, heparin andother glycosaminoglycans, such as chondroitin sulfates A or C, heparansulfates or dermatan sulfates. Exemplary assays for assessing HA bindingactivity are provided herein in Section D and in the Examples.

D. ASSAYS AND CLASSIFICATION

The methods provided herein are based on assaying the expression orlevels of hyaluronan (HA) in a sample or samples, such as a tissuesample or body fluid sample. The methods herein are based on bindingmethods using a hyaluronan binding protein companion diagnostic (HABP,such as a TSG-6-LM, multimer or variant thereof) for assessing,evaluating, determining, quantifying and/or otherwise specificallydetecting hyaluronan expression or levels in a sample. The assays can beperformed in vitro or in vivo. By comparisons to a control or referencesample or classifications based on a predetermined level, such valuescan be used for diagnosis or prognosis of a hyaluronan-associateddisease or condition, to predict responsiveness of a subject having ahyaluronan-associated disease or condition to a hyaluronan-degradingenzyme therapy, and/or to monitor or predict efficacy of treatment of asubject having a hyaluronan-associated disease or condition that hasbeen treated with a hyaluronan-degrading enzyme therapy. For example, asdescribed herein, it is found that HA levels and extent specifically areassociated with responsiveness to treatment with a hyaluronan-degradingenzyme, such as a hyaluornidase or modified hyaluronidase (e.g.PEGylated hyaluronidase such as PEGPH20).

In any of the above examples, the hyaluronan-associated diseases orconditions are diseases and conditions in which hyaluronan levels areelevated as cause, consequence or otherwise observed in the disease orcondition. Exemplary hyaluronan-associated diseases or conditions,include, but are not limited to, ones that are associated with highinterstitial fluid pressure, a cancer and in particular a hyaluronanrich cancer, edema, disc pressure, an inflammatory disease, and otherdiseases associated with hyaluronan. In some cases,hyaluronan-associated diseases and conditions are associated withincreased interstitial fluid pressure, decreased vascular volume, and/orincreased water content in a tissue, such as a tumor. In particular,hyaluronan-associated diseases and conditions, include, but are notlimited to, hyaluronan-rich cancers, for example, tumors, includingsolid tumors such as late-stage cancers, metastatic cancers,undifferentiated cancers, ovarian cancer, in situ carcinoma (ISC),squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer,non-small cell lung cancer, breast cancer, colon cancer and othercancers.

In one example, based on the levels or expression of hyaluronan, apatient or subject can be selected for treatment with an anti-hyaluronanagent (e.g. a hyaluronan-degrading enzyme). For example, a sample from asubject can be contacted with a hyaluronan-binding protein (HABP)companion diagnostic, such as TSG-6-LM, a multimer or variant thereof,and the binding of the HABP to the sample can be detected in order todetermine the amount of hyaluronan in the sample. Based on predeterminedselection or classification criteria as described herein, a patient canbe diagnosed with a hyaluronan-associated disease or condition, andhence selected for treatment of the disease or condition. Also, based onthe predetermined selection or classification criteria as describedherein, the methods herein can be used for prognosis of the subject.Depending on the course of the disease or condition, the dose, treatmentschedule and/or dosing regime of the therapeutic agent (e.g. ahyaluronan-degrading enzyme) can be optimized and adjusted accordingly.In particular examples herein, based on the predetermined selection orclassification criteria as described herein, a patient or subject can beselected for treatment that is predicted to be responsive to treatmentwith an anti-hyaluronan agent, for example a hyaluronan-degradingenzyme, such as a hyaluronidase or modified hyaluronidase (e.g. aPEGylated hyaluronidase such as PEGPH20). Hence, the method can be usedto predict the efficacy of treatment by an anti-hyaluronan agent, forexample a hyaluronan-degrading enzyme.

In examples of methods herein, the efficacy of the treatment by ananti-hyaluronan agent, for example a hyaluronan-degrading enzyme, can bedetermined by monitoring the expression or levels of hyaluronan over thecourse of treatment. Hence, the method is a post-treatment method ofmonitoring disease status and/or resolution, which information can beused to alter the course of treatment of a subject depending onindividualized status information. For example, a sample from a subjectcan be contacted with a hyaluronan-binding protein (HABP) companiondiagnostic, for example a TSG-6-LM, a multimer or variant thereof, andthe binding of the HABP to the sample can be detected in order todetermine the amount of hyaluronan in the sample. The expression orlevel of hyaluronan in the sample can be compared to a reference orcontrol sample in order to assess differences in hyaluronan levels orexpression. For example, elevated or accumulated hyaluronan levels in adiseased subject compared to a healthy or normal subject is indicativeof a hyaluronan-associated disease or condition (e.g. tumor or cancer)and the extent of the hyaluronan expression or levels correlates todisease aggressiveness. In such methods, the control or reference sampleis a sample from a healthy subject, is a baseline sample from thesubject prior to treatment with an anti-hyaluronan agent (e.g. ahyaluronan-degrading enzyme) (pre-treatment reference) or is a samplefrom a subject prior to the last dose of an anti-hyaluronan agent (e.g.a hyaluronan-degrading enzyme). For example, for monitoring patientresponse, the assay can be run at the initiation of therapy to establishbaseline (or predetermined) levels of hyaluronan in a sample (e.g.tissue or body fluid). The same sample (e.g. tissue or body fluid) isthen sampled using the same assay and the levels of hyaluronan comparedto the baseline or predetermined levels.

1. Assays for Measuring Hyaluronan

It is within the level of one of skill in the art to assess, quantify,determine and/or detect hyaluronan levels in a sample using an HABPcompanion diagnostic, such as TSG-6-LM, multimer (e.g. TSG-6LM-Fc) orvariant thereof, as described herein. Assays include in vitro or in vivoassays. Exemplary of binding assays that can be used to assess,evaluate, determine, quantify and/or otherwise specifically detecthyaluronan expression or levels in a sample include, but are not limitedto, solid phase binding assays (e.g. enzyme linked immunosorbent assay(ELISA)), radioimmunoassay (RIA), immunoradiometric assay, fluorescencceoassay, chemiluminescent assay, bioluminescent assay, western blot andhistochemistry methods, such as immunohistochemistry (IHC) or pseudoimmunohistochemistry using a non-antibody binding agent. In solid phasebinding assay methods, such as ELISA methods, for example, the assay canbe a sandwich format or a competitive inhibition format. In otherexamples, in vivo imaging methods can be used.

a. Histochemical and Immunohistochemical Methods

The methods of assessing hyaluronan accumulation are based on theability of an HABP companion diagnostic to bind to HA in a sample, forexample a tissue or cell sample, such that the amount of the HABPcompanion diagnostic that binds correlates with amount of HA in thesample. Any HABP companion diagnostic provided herein can be used todetect HA using tissue staining methods known to one of skill in theart, including but not limited to, cytochemical or histochemicalmethods, such as immunohistochemistry (IHC) or histochemistry using anon-antibody binding agent (e.g. pseudo immunohistochemistry). Suchhistochemical methods permit quantitative or semi-quantitative detectionof the amount of HABP that binds to HA in a sample, such as a tumortissue sample. In such methods, a tissue sample can be contacted with anHABP reagent provided herein, and in particular one that is detectablylabeled or capable of detection, under conditions that permit binding totissue- or cell-associated HA.

A sample for use in the methods provided herein as determined byhistochemistry can be any biological sample that can be analyzed for itsHA levels, such as a tissue or cellular sample. For example, a tissuesample can be solid tissue, including a fresh, frozen and/or preservedorgan or tissue sample or biopsy or aspirate, or cells. In someexamples, the tissue sample is tissue or cells obtained from a solidtumor, such as primary and metastatic tumors, including but not limitedto, breast, colon, rectum, lung, stomach, ovary, cervix, uterus, testes,bladder, prostate, thyroid and lung cancer tumors. In particularexamples, the sample is a tissue sample from a cancer is a late-stagecancer, a metastatic cancer, undifferentiated cancer, ovarian cancer, insitu carcinoma (ISC), squamous cell carcinoma (SCC), prostate cancer,pancreatic cancer, non-small cell lung cancer, breast cancer, or coloncancer. In other examples, the tissue sample contains cells from primaryor cultured cells or cell lines. Cells may be have various states ofdifferentiation, and may be normal, pre-cancerous or cancerous, may befresh tissues, diespersed cells, immature cells, including stem cells,cells of intermediate maturity and fully matured cells. Typically, thecells selected for use in the methods provided herein are cancer cells.

When the tumor is a solid tumor, isolation of tumor cells is typicallyachieved by surgical biopsy. Biopsy techniques that can be used toharvest tumor cells from a subject include, but are not limited to,needle biopsy, CT-guided needle biopsy, aspiration biopsy, endoscopicbiopsy, bronchoscopic biopsy, bronchial lavage, incisional biopsy,excisional biopsy, punch biopsy, shave biopsy, skin biopsy, bone marrowbiopsy, and the Loop Electrosurgical Excision Procedure (LEEP).Typically, a non-necrotic, sterile biopsy or specimen is obtained thatis greater than 100 mg, but which can be smaller, such as less than 100mg, 50 mg or less, 10 mg or less or 5 mg or less; or larger, such asmore than 100 mg, 200 mg or more, or 500 mg or more, 1 gm or more, 2 gmor more, 3 gm or more, 4 gm or more or 5 gm or more. The sample size tobe extracted for the assay can depend on a number of factors including,but not limited to, the number of assays to be performed, the health ofthe tissue sample, the type of cancer, and the condition of the subject.The tumor tissue is placed in a sterile vessel, such as a sterile tubeor culture plate, and can be optionally immersed in an appropriatemedium.

Tissue obtained from the patient after biopsy is often fixed, usually byformalin (formaldehyde) or glutaraldehyde, for example, or by alcoholimmersion. For histochemical methods, the tumor sample can be processedusing known techniques, such as dehydration and embedding the tumortissue in a paraffin wax or other solid supports known to those of skillin the art (see Plenat et al., (2001) Ann Pathol January 21(1):29-47),slicing the tissue into sections suitable for staining, and processingthe sections for staining according to the histochemical staining methodselected, including removal of solid supports for embedding by organicsolvents, for example, and rehydration of preserved tissue. Thus,samples for use in the methods herein can contain compounds that are notnaturally present in a tissue or cellular sample, including for example,preservatives, anticoagulants, buffers, fixatives, nutrients andantibiotics.

In exemplary methods to select a subject for treatment with ahyaluronan-degrading enzyme, harvesting of the tumor tissue is generallyperformed prior to treatment of the subject with a hyaluronan-degradingenzyme. In exemplary methods of monitoring therapy of a tumor with ahyaluronan-degrading enzyme, harvesting of the tumor tissue from thesubject can be performed before, during or after the subject hasreceived one or more treatments with a hyaluronan-degrading enzyme.

Assays for use in the methods provided herein are those in which HApresent in the sample is detected using histochemistry orimmunohistochemistry. Histochemistry (HC) is a staining method based onenzymatic reactions using a binding partner, such as an antibody (e.g.monoclonal or polyclonal antibodies) or other binding partner, to detectcells or specific proteins such as tissue antigens, or biomarkers, forexample, HA. For example, histochemistry assays for use in the methodsherein include those where an HABP is used as a binding partner todetect HA associated with cells or tissues. Typically, histochemistryprotocols include detection systems that make the presence of themarkers visible, to either the human eye or an automated scanningsystem, for qualitative or quantitative analyses. In a direct HC assay,binding is determined directly upon binding of the binding partner (e.g.first antibody) to the tissue or biomarker due to the use of a labeledreagent. In an indirect HC assay, a secondary antibody or second bindingpartner is necessary to detect the binding of the first binding partner,as it is not labeled.

In such methods, generally a slide-mounted tissue sample is stained witha labeled binding reagent (e.g. labeled HABP) using commonhistochemistry techniques. Thus, in exemplary HC methods providedherein, the HABP companion diagnostics are modified to contain a moietycapable of being detected (as described in Section 3C above). In someexamples, the HABP companion diagnostics are conjugated to smallmolecules, e.g., biotin, that are detected via a labeled binding partneror antibody. In some examples, the IHC method is based on staining withan HABP protein that is detected by enzymatic staining with horseradishperoxidase. For example, the HABP can be biotinylated and detected withavidin or streptavidin conjugated to detectable protein, such asstreptavidin-horseradish peroxidase (see Example 6 below). In otherexamples, the HABP companion diagnostics are conjugated to detectableproteins which permit direct detection, such as, for example, HABPcompanion diagnostics conjugated to a fluorescent protein,bioluminescent protein or enzyme. Various enzymatic staining methods areknown in the art for detecting a protein of interest. For example,enzymatic interactions can be visualized using different enzymes such asperoxidase, alkaline phosphatase, or different chromogens such as DAB,AEC or Fast Red. In other examples, the HABP companion diagnostics areconjugated to peptides or proteins that can be detected via a labeledbinding partner or antibody.

In other examples, HA is detected by HC methods using an HABP companiondiagnostic provided herein where the HABP companion diagnostics aredetected by labeled secondary reagents, such as labeled antibodies thatrecognize one or more epitopes of the HABPs, HABP link domains, or HAbinding fragments thereof. In other examples, HABP companion diagnosticsare detected using an anti-HABP antibody. For detecting an HABP, anyanti-HABP antibody can be used so long as it binds to the HABP, HABPlink domain, or HA binding fragment thereof used to detect HA. Forexample, for detecting TSG-6 or a TSG-6-LM, an anti-TSG-6 link modulemonoclonal antibody can be used, such as antibodies designated A38 andQ75 (see, Lesley et al. (2002) J Biol Chem 277:26600-26608). Theanti-HABP antibodies can be labeled for detection or can be detectedwith a secondary antibody that binds the first antibody. The selectionof an appropriate anti-HABP antibody is within the level of one of skillin the art.

The resulting stained specimens are each imaged using a system forviewing the detectable signal and acquiring an image, such as a digitalimage of the staining Methods for image acquisition are well known toone of skill in the art. For example, once the sample has been stained,any optical or non-optical imaging device can be used to detect thestain or biomarker label, such as, for example, upright or invertedoptical microscopes, scanning confocal microscopes, cameras, scanning ortunneling electron microscopes, canning probe microscopes and imaginginfrared detectors. In some examples, the image can be captureddigitally. The obtained images can then be used for quantitatively orsemi-quantitatively determining the amount of HA in the sample. Variousautomated sample processing, scanning and analysis systems suitable foruse with immunohistochemistry are available in the art. Such systems caninclude automated staining and microscopic scanning, computerized imageanalysis, serial section comparison (to control for variation in theorientation and size of a sample), digital report generation, andarchiving and tracking of samples (such as slides on which tissuesections are placed). Cellular imaging systems are commerciallyavailable that combine conventional light microscopes with digital imageprocessing systems to perform quantitative analysis on cells andtissues, including immunostained samples. See, e.g., the CAS-200 system(Becton, Dickinson & Co.). In particular, detection can be made manuallyor by image processing techniques involving computer processors andsoftware. Using such software, for example, the images can beconfigured, calibrated, standardized and/or validated based on factorsincluding, for example, stain quality or stain intensity, usingprocedures known to one of skill in the art (see e.g. published U.S.patent Appl. No. US20100136549).

The image can be quantitatively or semi-quantitatively analyzed andscored based on staining intensity of the sample. Quantitative orsemi-quantitative histochemistry refers to method of scanning andscoring samples that have undergone histochemistry, to identify andquantitate the presence of a specified biomarker, such as an antigen orother protein (e.g. HA). Quantitative or semi-quantitative methods canemploy imaging software to detect staining densities or amount ofstaining or methods of detecting staining by the human eye, where atrained operator ranks results numerically. For example, images can bequantitatively analyzed using a pixel count algorithms (e.g. AperioSpectrum Software, Automated QUantitatative Analysis platform (AQUA®platform), and other standard methods that measure or quantitate orsemi-quantitate the degree of staining; see e.g. U.S. Pat. No.8,023,714; U.S. Pat. No. 7,257,268; U.S. Pat. No. 7,219,016; U.S. Pat.No. 7,646,905; published U.S. Pat. Appl. Nos. US20100136549 and20110111435; Camp et al. (2002) Nature Medicine, 8:1323-1327; Bacus, etal. (1997) Analyt Quant Cytol Histol, 19:316-328). A ratio of strongpositive stain (such as brown stain) to the sum of total stained areacan be calculated and scored.

Using histochemical, such as immunohistochemical or pseudoimmunohistochemical methods, the amount of HA detected is quantified andgiven as a percentage of HA positive pixels and/or a score. For example,the amount of HA detected in the sample can be quantified as apercentage of HA positive pixels. In some examples, the amount of HApresent in a sample is quantified as the percentage of area stained,e.g., the percentage of HA positive pixels. For example, a sample canhave at least or about at least or about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more HApositive pixels as compared to the total staining area.

In some examples, a score is given to the sample that is a numericalrepresentation of the intensity or amount of the histochemical stainingof the sample, and represents the amount of target biomarker (e.g., HA)present in the sample. Optical density or percentage area values can begiven a scaled score, for example on an integer scale, for example,0-10, 0-5, or 0-3. In particular examples, the amount of hyaluronan in asample is classified on a scale of 0-3, e.g. 0, HA⁺¹, HA⁺², and HA⁺³.The amount of HA present is relative to the percentage of HA pixels,that is, low percentages of HA pixels indicates a low level of HAwhereas high percentages of HA pixels indicate high levels of HA. Scorescan correlated with percentages of HA positive pixels, such that thepercentage area that is stained is scored as 0, HA⁺¹, HA⁺², and HA⁺³,representing no staining, less than 10% staining, 10-25% staining ormore than 25% staining respectively. For example, if the ratio (e.g.strong pixel stain to total stained area) is more than 25% the tumortissue is scored as HA⁺³, if the ratio is 10-25% of strong positivestain to total stain the tumor tissue is scored as HA⁺², if the ratioless than 10% of strong positive stain to total stain the tumor tissueis scored as HA⁺¹, and if the ratio of strong positive stain to totalstain is 0 the tumor tissue is scored as 0. A score of 0 or HA⁺¹indicates low levels of HA in the tested sample, whereas a score of HA⁺²or HA indicates higher levels of HA in the tested samples.

b. Solid Phase Binding Assays

The methods of assessing hyaluronan accumulation are based on theability of an HABP companion diagnostic to bind to HA in a sample suchthat the amount of the HABP companion diagnostic that binds correlateswith amount of HA in the sample.

In particular solid-phase binding assays can be used. Exemplary ofbinding assays that can be used to assess, evaluate, determine, quantifyand/or otherwise specifically detect hyaluronan expression or levels ina sample include, but are not limited to, enzyme linked immunosorbentassay (ELISA), radioimmunoassay (RIA), immunoradiometric assay,fluroassay, chemiluminescent assay, bioluminescent assay. For example,an HABP companion diagnostic provided herein can detect HA using anybinding assay known to one of skill in the art, including but notlimited to, enzyme-linked immunosorbent assay (ELISA) or other similarimmunoassay, including a sandwich ELISA or competitive ELISA assay.Exemplary methods provided herein include ELISA based methods forquantitative or semi-quantitative detection of the amount of HABP thatbinds to HA in a sample, such as a tumor tissue sample or fluid samplefrom a subject having a tumor or suspected of having a tumor. The use ofsolid phase binding assays can be used when HA is detected in a bodilyfluid.

As described herein, patients that exhibit high levels of hyaluronanproduction in the tumor tissue also exhibit high levels of hyaluronan inblood. Accordingly, the methods provided herein encompass methods ofpredicting the responsiveness of a subject to treatment with ahyaluronan-degrading enzyme, to select subjects for treatment with ahyaluronan-degrading enzyme, or to monitor treatment with a hyaluronandegrading enzyme, including assessing the accumulation of hyaluronan ina fluid sample from a patient having a tumor or a patient suspected ofhaving a tumor.

Fluid samples for analysis of HA production in an HA-associated disease,such as cancer, include but are not limited to serum, urine, plasma,cerebrospinal fluid, and lymph. The subject can have or be suspected ofhaving a cancer, such as a primary and metastatic tumors, in breast,colon, rectum, lung, stomach, ovary, cervix, uterus, testes, bladder,prostate, thyroid, lung cancer. In particular examples, the cancer is alate-stage cancer, a metastatic cancer, undifferentiated cancer, ovariancancer, in situ carcinoma (ISC), squamous cell carcinoma (SCC), prostatecancer, pancreatic cancer, non-small cell lung cancer, breast cancer, orcolon cancer.

In exemplary methods to predict the responsiveness of subject totreatment with a hyaluronan-degrading enzyme or to select subjects fortreatment with a hyaluronan-degrading enzyme, collection of a fluidsample from a subject is generally performed prior to treatment of thesubject with a hyaluronan-degrading enzyme. In exemplary methods ofmonitoring therapy of a tumor with a hyaluronan-degrading enzyme,collection of the fluid sample from a subject can be performed before,during or after the subject has received one or more treatments with ahyaluronan-degrading enzyme. Harvesting of the fluid sample also can beperformed before, during, or after the subject has undergone one or morerounds of anti-cancer therapy, such as radiation and/or chemotherapytreatment.

The fluid sample then can be assessed for the presence or amount of HAusing a solid-phase binding assay. Solid-phase binding assays can detecta substrate (e.g. HA) in a fluid sample by binding of the substrate to abinding agent that is fixed or immobilized to a solid surface. Asubstrate specific antibody or binding protein (e.g. an HABP providedherein), coupled to detectable label (e.g. an enzyme), is applied andallowed to bind to the substrate. Presence of the antibody or boundprotein is then detected and quantitated. Detection and quantitationmethods include, but are not limited to, colorimetric, fluorescent,luminescent or radioactive methods. The choice of detection method isdependent on the detectable label used. In some examples, a colorimetricreaction employing the enzyme coupled to the antibody. For example,enzymes commonly employed in this method include horseradish peroxidaseand alkaline phosphatase. The amount of substrate present in the sampleis proportional to the amount of color produced. A substrate standard isgenerally employed to improve quantitative accuracy. The concentrationof HA in a sample can be calculated by interpolating the data to astandard curve. The amount of HA can be expressed as a concentration offluid sample.

In an exemplary method, an HABP reagent that is generally unlabeled isfirst immobilized to a solid support (e.g. coated to wells of amicrotiter plate), followed by incubation with a fluid sample containingHA (e.g. serum or plasma) to capture HA. After washing the fluid samplewith an appropriate buffer, bound HA can be detected. In some examplesto detect the bound HA, a second HABP that is the same or different thanthe immobilized HABP and that is labeled (labeled HABP), such as abiotinylated HABP, is used to bind to the HA on the plate. Followingremoval of the unbound labeled HABP, the bound labeled HABP is detectedusing a detection reagent. For example, biotin can be detected using anavidin detection reagent. In some examples, the HABP bound to the plateis different from the HABP used for detection. In other examples, theHABP bound to the plate and the HABP for detection are the same. Inother examples to detect the bound HA, bound HA is detected by additionof HABP and subsequent addition of an anti-HABP antibody. For example,for detecting TSG-6 or a TSG-6-LM, an anti-TSG-6 link module monoclonalantibody can be used, such as antibodies designated A38 and Q75 (see,Lesley et al. (2002) J Biol Chem 277:26600-26608). The anti-HABPantibodies can be labeled for detection or can be detected with asecondary antibody that binds the first antibody. In yet other examplesto detect the bound HA, bound HA is directly detected with an anti-HAantibody. Anti-HA antibodies are well known to one of skill in the art,and include, for example, a sheep anti-hyaluronic acid polyclonalantibody (e.g., Abcam #53842 or #93321).

c. In Vivo Imaging Assays

In some examples herein, the amount of HA is detected using in vivoimaging methods. In such methods, the HABP, such as a TSG-6-LM, multimer(e.g. TSG-6LLM-Fc) or variant thereof, is conjugated to a detectablemoiety or moiety that is capable of detection by an imaging method.Exemplary imaging methods include, but are not limited to, fluorescenceimaging, X-rays, magnetic resonance methods, such as magnetic resonanceimaging (MRI) and magnetic resonance spectroscopy (MRS), and tomographicmethods, including computed tomography (CT), computed axial tomography(CAT), electron beam computed tomography (EBCT), high resolutioncomputed tomography (HRCT), hypocycloidal tomography, positron emissiontomography (PET), single-photon emission computed tomography (SPECT),spiral computed tomography and ultrasonic tomography. For example, forfluorescence imaging, fluorescent signals can be analyzed using afluorescent microscope or fluorescence stereomicroscope. Also, a lowlight imaging camera also can be used.

In particular, the HABP, such as a TSG-6-LM, multimer (e.g. TSG-6LLM-Fc)or variant thereof, is labeled or conjugated with a moiety that providesa signal or induces a signal that is detectable in vivo, when imaged,such as, but not limited to, magnetic resonance imaging (MRI),single-photon emission computed tomography (SPECT), positron emissiontomography (PET), scintigraphy, gamma camera, α β+ detector, a γdetector, fluorescence imaging and bioluminescence imaging. Exemplaryimaging/monitoring methods include any of a variety magnetic resonancemethods such as magnetic resonance imaging (MRI) and magnetic resonancespectroscopy (MRS), and also include any of a variety of tomographicmethods including computed tomography (CT), computed axial tomography(CAT), electron beam computed tomography (EBCT), high resolutioncomputed tomography (HRCT), hypocycloidal tomography, positron emissiontomography (PET), gamma rays (after annihilation of a positron and anelectron in PET scanning), single-photon emission computed tomography(SPECT), spiral computed tomography and ultrasonic tomography. Otherexemplary imaging methods include low-light imaging, X-rays, ultrasoundsignal, fluorescence absorption and bioluminescence. In addition, theproteins can be labeled with light-emitting or other electromagneticspectrum-emitting compounds, such as fluorescent compounds or molecules.Detection can be effected by detecting emitted light or other emittedelectromagnetic radiation.

Detectable labels include reagents with directly detectable elements(e.g. radiolabels) and reagents with indirectly detectable elements(e.g. a reaction product). Section C.3.c also describes detectablelabels. Examples of detectable labels include radioisotopes,bioluminescent compounds, chemiluminescent compounds, fluorescentcompounds, metal chelates and enzymes. A detectable label can beincorporated into an HABP by chemical or recombinant methods.

Labels appropriate for X-ray imaging are known in the art, and include,for example, Bismuth (III), Gold (III), Lanthanum (III) or Lead (II); aradioactive ion, such as 67Copper, 67Gallium, 68Gallium, 111Indium,113Indium, 123Iodine, 125Iodine, 131Iodine, 197Mercury, 203Mercury,186Rhenium, 188Rhenium, 97Rubidium, 103Rubidium, 99Technetium or90Yttrium; a nuclear magnetic spin-resonance isotope, such as Cobalt(II), Copper (II), Chromium (III), Dysprosium (III), Erbium (III),Gadolinium (III), Holmium (III), Iron (II), Iron (III), Manganese (II),Neodymium (III), Nickel (II), Samarium (III), Terbium (III), Vanadium(II) or Ytterbium (III); or rhodamine or fluorescein.

Contrast agents are used for magnetic resonance imaging. Exemplarycontrast agents include iron, gold, gadolinium and gallium. Labelsappropriate for magnetic resonance imaging are known in the art, andinclude, for example, fluorine, gadolinium chelates, metals and metaloxides, such as for example, iron, gallium, gold, gadolinium, magnesium,¹H, ¹⁹F, ¹³C, and ¹⁵N labeled compounds. Use of chelates in contrastagents is known in the art. Labels appropriate for tomographic imagingmethods are known in the art, and include, for example, β-emitters suchas ¹¹C, ¹³N, ¹⁵O or ⁶⁴Cu or (b) γ-emitters such as ¹²³I. Other exemplaryradionuclides that can, be used, for example, as tracers for PET include⁵⁵Co, ⁶⁷Ga, ⁶⁸Ga, ⁶⁰Cu(II), ⁶⁷Cu(II), ⁹⁹Tc, ⁵⁷Ni, ⁵²Fe and ¹⁸F. Thereagent, such as TSG-6 or the FC portion thereof can be conjugated to asuitable label and/or the protein can include a radiolabel in itsconstituent molecules.

An exemplary list of radionuclides useful for the imaging methodsprovided herein includes, for example, ¹¹Carbon, ¹¹Fluorine, ¹³Carbon,¹³Nitrogen, ¹⁵Nitrogen, ¹⁵Oxygen, ¹⁸Flourine, ¹⁹Flourine, ²⁴Sodium,³²Phosphate, ⁴²Potassium, ⁵¹Chromium, ⁵⁵Iron, ⁵⁹Iron, ⁵⁷Cobalt,⁶⁰Cobalt, ⁶⁴Copper, ⁶⁷Gallium, ⁶⁸Gallium, ⁷⁵Selenium, ⁸¹Krypton,⁸²Rubidium, ⁸⁹Strontium, ⁹²Strontium, ⁹⁰Yttrium, ⁹⁹Technetium,¹⁰³Palladium, ¹⁰⁶Ruthenium, ¹¹¹Indium, ¹¹⁷Lutetium, ¹²³Iodine,¹²⁵Iodine, ¹³¹Iodine, ¹³³Xenon, ¹³⁷Cesium, ¹⁵³Samarium, ¹⁵³Gadolinium,¹⁶⁵Dysprosium, ¹⁶⁶Holmium, ¹⁶⁹Ytterbium, ¹⁷⁷Leutium ¹⁸⁶Rhenium,¹⁸⁸Rhenium, ¹⁹²Iridium, ¹⁹⁸Gold, ²⁰¹Thallium, ²¹¹Astatine, ²¹²Bismuthand ²¹³Bismuth. One of skill in the art can alter the parameters used indifferent imaging methods (MRI, for example) in order to visualizedifferent radionuclides/metals.

Fluorescent labels also can be used. These include fluorescent proteins,fluorescent probes or fluorescent substrate. For example, fluorescentproteins can include, but are not limited to, fluorescent proteins suchas green fluorescent protein (GFP) or homologs thereof or RFP;fluorescent dyes (e.g., fluorescein and derivatives such as fluoresceinisothiocyanate (FITC) and Oregon Green®, rhodamine and derivatives(e.g., Texas red and tetramethyl rhodamine isothiocyanate (TRITC)),biotin, phycoerythrin, AMCA, Alexa Fluor®, Li-COR®, CyDyes® or DyLight®Fluors); tdTomato, mCherry, mPlum, Neptune, TagRFP, mKate2, TurboRFP andTurboFP635 (Katushka). The fluorescent reagent can be chosen based onuser desired excitation and emission spectra. Fluorescent substratesalso can be used that result in fluorescent cleavage products.

The in vivo imaging methods can be used in the diagnosis ofHA-associated tumors or cancers. Such a technique permits diagnosiswithout the use of biopsy. In vivo imaging methods based on the extentor level of binding of an HABP to a tumor also can be used for prognosesto cancer patients. The in vivo imaging methods also can be used todetect metastatic cancers in other parts of the body or circulatingtumor cells (CTCs). It is within the level of one of skill in the art toascertain background levels of hyaluronan in tissues other than tumors.Hyaluronan-expressing tumors will have higher levels of signal thanbackground tissues. In some examples, threshold criteria can bedetermined by comparisons to signal detected in normal or healthysubjects.

2. Classification of Subjects

Once the amount of hyaluronan in the sample is determined, the amountcan be compared to a control or threshold level. The control orthreshold level is generally a predetermined threshold level or amountthat is indicative of a hyaluronan-associated disease or condition (e.g.a tumor or cancer). Such level or amount can be empirically determinedby one skilled in the art. It is understood that the particularpredetermined selection or classification criteria for the methodsherein are dependent on the particular assay that is used to detecthyaluronan and the particular sample that is being tested. It is withinthe level of one of skill in the art to determine if an assay iscompatible with testing a particular sample. Generally, in vitro solidphase assays are used for testing body fluid samples. Solid phase assayssuch as histochemistry or immunohistochemistry are generally used fortesting tissue samples. It also is understood that in methods involvingcomparisons to a predetermined level or amount or to a control orreference sample that the references are made with the same type ofsample and using the same assay and HABP reagent (including the samedetectable moiety and detecting method).

For example, the predetermined threshold level can be determined basedon the level or amount of the marker in a reference or control sample,such as the median or mean level or amount of the marker in a populationof subjects, in order to assess differences in levels or expression. Inone example, the predetermined threshold level can represent the mean ormedian level or amount of hyaluronan in a sample from a healthy subjector a subject known to have a hyaluronan-associated disease or condition(e.g. a tumor or cancer). In one embodiment, the predetermined level oramount of hyaluronan from a normal tissue or bodily fluid sample is themean level or amount observed in normal samples (e.g., all normalsamples analyzed). In another embodiment, the level or amount ofhyaluronan from a normal tissue or bodily fluid sample is the medianvalue for the level or amount observed in normal samples. Thepredetermined threshold level also can be based on the level or amountof hyaluronan in a cell line or other control sample (e.g. tumor cellline). As described below, these predetermined values can be determinedby comparison or knowledge of HA levels in a corresponding normal sampleas determined by the same assay of detection and using the same HABPreagent.

The reference or control sample can be another tissue, cell or bodyfluid, such as a normal tissue, cell or body fluid, for example, atissue, cell or body fluid that is analogous to the sample being tested,but isolated from a different subject. The control or reference subjectcan be a subject or a population of subjects that is normal (i.e. doesnot have a disease or condition), a subject that has a disease but doesnot have the type of disease or condition that the subject being testedhas or is suspected of having, for example, a subject that does not havea hyaluronan-associated disease or condition (e.g. a tumor or cancer),or an analogous tissue from another subject that has a similar diseaseor condition, but whose disease is not as severe and/or expressesrelatively less hyaluronan. For example, when the cell, tissue or fluidbeing tested is a subject or a population of subjects having a cancer,the level or amount of the marker can be compared to the level or amountof the marker in a tissue, cell or fluid from a subject having a lesssevere cancer, such as an early stage, differentiated or other type ofcancer. In another example, a control or reference sample is a fluid,tissue, extract (e.g. cellular or nuclear extract), nucleic acid orpeptide preparation, cell line, biopsy, standard or other sample, with aknown amount or relative amount of hyaluronan, such as a sample, forexample a tumor cell line, known to express relatively low levels of HA,such as exemplary tumor cell lines that express low levels of HA, forexample, the HCT 116 cell line, the HT29 cell line, the NCI H460 cellline, the DU145 cell line, the Capan-1 cell line, and tumors from tumormodels generated using such cell lines.

In any method herein, the level(s) of hyaluronan in samples fromsubjects suspected or known to have a hyaluronan-associated disease orcondition (e.g., cancer) can be determined concurrently with thedetermination of level(s) of hyaluronan in reference or normal tissues.Alternatively, the levels of hyaluronan in samples from subjectssuspected or known to have a hyaluronan-associated disease or condition(e.g. cancer) can be compared to the level(s) of hyaluronan previouslydetermined in normal tissue or bodily fluid. Thus, the level ofhyaluronan in normal or healthy samples or other reference samplesemployed in any detection, comparison, determination, or evaluation canbe a level or amount determined prior to any detection, determination,or evaluation of the level or amount of hyaluronan in a sample from ahuman patient.

The level or amount of hyaluronan of is determined and/or scored andcompared to predetermined phenotypes of Hyaluronan associated withdisease. It is within the level of one of skill in the art to determinethe threshold level for disease diagnosis depending on the particulardisease, the assay being used for detection of the hyaluronan and/or theHABP detection reagent being used. It is within the level of one ofskill in the art to determine the threshold level of the hyaluronan forclassifying responsiveness to treatment with an anti-hyaluronan agent(e.g. a hyaluronan-degrading enzyme). Exemplary methods forstratification of tumor samples or bodily fluid samples for diagnosis,prognosis or selection of subjects for treatment are provided herein.

It is understood that the particular change, e.g. increase in ordecrease of hyaluronan is dependent on the assay used. In an ELISA, thefold increase or decrease in absorbance at a particular wavelength or inquantity of protein (e.g. as determined by using a standard curve) canbe expressed relative to a control. In a PCR assay, such as RT-PCR,expression levels can be compared to control expression levels (e.g.expressed as fold change) using methods known to those in the art, suchas using standards.

In particular examples of the methods herein, a subject is selected as acandidate for therapy with an anti-hyaluronan agent if the amount ofhyaluronan is determined to be elevated in the sample. For example,elevated or accumulated hyaluronan levels in a diseased subject comparedto a healthy or normal subject is indicative of a hyaluronan-associateddisease or condition (e.g. tumor or cancer). The hyaluronan can beelevated 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold or more. Thus, in examples of themethods herein, when the amount of hyaluronan in a sample from a subjectis being tested, detection of the marker can be determining that theamount of HA in the sample (e.g. cancerous cell, tissue or fluid) fromthe subject is elevated compared to a predetermined level or amount orcontrol sample. In one example, the subject is determined to have aHyaluronan-associated disease or condition if the amount of HA in thetissue, cell or fluid is elevated at or about 0.5-fold, 1-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, or more, comparedto the predetermined level or amount or control sample.

A subject can be selected as a candidate for therapy with ananti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme) based on thelevel or amount of hyaluronan in a sample (e.g. a bodily fluid or otherfluid) from the subject. HA greater than 0.010 μg/mL, 0.015 μg/mL, andgenerally greater than 0.02 μg/mL, 0.03 μg/mL, 0.04 μg/mL, 0.05 μg/mL,0.06 μg/mL or higher correlates to the presence of a tumor or cancer.Using such methods, in exemplary methods provided herein, a subject canbe selected for treatment with a an anti-hyaluronan agent (e.g.hyaluronan-degrading enzyme) if the concentration of HA in the fluidsample, such as a serum sample, contains HA levels greater than 0.010μg/mL, 0.015 μg/mL, and generally greater than 0.02 μg/mL, 0.03 μg/mL,0.04 μg/mL, 0.05 μg/mL, 0.06 μg/mL or higher.

A subject can be selected as a candidate for therapy with a ananti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme) based on thelevel or amount of hyaluronan in a cell or tissue sample. In such anexample, if the level is indicative of disease, then the patient isdiagnosed with a hyaluronan-associated disease or condition. Forexample, using immunohistochemistry methods of tumor tissues a score ofHA⁺² or HA⁺³ can be determinative of disease. For example, a percentageof staining of HA over total tumoral area of greater than 10%, such as10 to 25%, or greater than 25% is indicative of disease. In the methodsherein, a subject is selected for treatment with an anti-hyaluronanagent (e.g. a hyaluronan-degrading enzyme) if the scaled score of thesample is an HA⁺² or HA⁺³ sample. For example, a high score, e.g., HA⁺³,indicates the subject has an HA-rich tumor indicative of the presence ofa tumor that would benefit from treatment with an anti-hyaluronan agent(e.g. a hyaluronan-degrading enzyme) and thus is a candidate for therapywith an anti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme). Inother examples, a subject can be selected for treatment with ananti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme) based on thepercentage of staining, for example, if the degree of HA staining is10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or more of the total staining area, and generally atleast 25% or more.

Efficacy of treatment with an anti-hyaluronan agent (e.g. ahyaluronan-degrading enzyme) or responsiveness to treatment also can bemonitored by comparing the level or amount of hyaluronan in a subjectover time. Changes in the level or amount of hyaluronan can be used tooptimize dosing or scheduling of treatment with an anti-hyaluronan agent(e.g. a hyaluronan-degrading enzyme). In the method, the level of HAexpression in samples, in particular as assessed in tumor tissues (e.g.via immunohistochemistry or other similar method), from treated subjectsare compared to a predetermined level of HA expression. For purposes ofmonitoring treatment after administration of a hyaluronan-degradingenzyme, in particular one with an extended half-life (e.g. PEGPH20), thesample that is monitored is not a bodily fluid in which systemic levelsof enzyme can be present.

For purposes of monitoring treatment, the predetermined level of HA canbe from a normal or healthy subject, a baseline HA value prior totreatment, the prior measured HA level in the same subject at an earliertime after treatment, or a classification or stratification of HA levelsknown to be associated with disease progression or regression. Forexample, if the hyaluronan level is about the same as or below (ordecreased) as compared reference or control sample, the treatment islikely efficacious and the treatment can be continued or discontinued oraltered. For example, the predetermined level of HA can be an HA levelfrom a normal or healthy tissue sample, and if the level of HA measuredin the subject after treatment is higher than the normal HA levels, thentreatment is resumed or continued. For example, the predetermined levelof HA can be HA levels as determined from a baseline HA value prior totreatment, and the course of treatment determined accordingly. Forexample, if the level of HA is the same as baseline HA, then treatmentis continued or resumed; if the level of HA is higher than baseline HA,then treatment is continued or resumed or treatment is accelerated orincreased (e.g. by increasing the dosage of hyaluronan-degrading enzymeor increasing the dose schedule in a dosage regimen cycle); if the levelof HA is less than baseline HA, then treatment is continued or resumed,terminated or is reduced or decreased (e.g. by decreasing the dosage ofhyaluronan-degrading enzyme or decreasing the dose schedule in a dosageregimen cycle). In a further example, the predetermined level of HA canbe an HA level as determined in a prior measurement in an earlier courseof treatment of the same subject. For example, if the level of HA is thesame as the earlier measured HA, then treatment is continued or resumed;if the level of HA is higher than the earlier measured HA, thentreatment is continued or resumed or treatment is accelerated orincreased (e.g. by increasing the dosage of hyaluronan-degrading enzymeor increasing the dose schedule in a dosage regimen cycle); if the levelof HA is less than the earlier measured HA, then treatment is continuedor resumed, terminated or is reduced or decreased (e.g. by decreasingthe dosage of hyaluronan-degrading enzyme or decreasing the doseschedule in a dosage regimen cycle).

In the monitoring methods or methods of determining efficacy oftreatment, the particular therapy can be altered during the course oftreatment to maximize individual response. Dosing and scheduling oftreatment can be modified in response to changing levels. Combinationtherapy using other anti-cancer agents also can be employed in suchtreatment methods. It is within the level of the skill of the treatingphysician to determine the exact course of treatment. For example, thetreatment can be altered, such that the dosing amount, schedule (e.gfrequency of administration), or regime is adjusted accordingly, such asdiscontinued, decreased or made less frequent, or combined with anothertreatment for the disease or condition. On the other hand, if thehyaluronan level is above a compared reference or control sample, thepatient is likely not responding to the treatment. In such instances,the particular nature and type of anti-hyaluronan agent (e.g.hyaluronan-degrading enzyme) or combination therapy can be modified orchanged. In other instances, the dosing, amount, schedule and/or regimecan be adjusted accordingly, such as increased or made more frequent. Itis within the level of the treating physician to determine the exactcourse of treatment.

For purposes of monitoring efficacy of treatment, predetermined levelsor amounts of hyaluronan can be empirically determined, whereby thelevel or amount indicates that the treatment is working. Thesepredetermined values can be determined by comparison or knowledge of HAlevels in a corresponding normal sample or samples of disease subjectsas determined by the same assay of detection and using the same HABPreagent. For example, high levels of HA as assessed byimmunohistochemistry methods using a quantitative score scheme (e.g.HA⁺³) or percentage of tumor staining for hyaluronan of greater than 25%correlate to the existence of malignant disease across a range of cancertypes, and indicate that a patient is not responding to treatment. Inanother example, HA levels in bodily fluid such as plasma of greaterthan 0.015 μg/mL, and generally greater than 0.02 μg/mL, such as 0.03μg/mL, 0.04 μg/mL, 0.05 μg/mL or 0.06 μg/mL HA, is associated withadvanced disease stage. On the other hand, a subject is likelyresponding to treatment if the scaled score of the sample is less thanan HA⁺² or HA⁺³ or the percentage of HA staining is less than 25%, 20%,15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or less. A subject is likelyresponding to treatment if the HA level in bodily fluid such as plasmais less than 0.03 μg/mL, 0.02 μg/mL, 0.01 μg/mL or less.

In the methods herein, the comparison to a predetermined level or tolevels of a control or reference sample can be determined by any methodknown of skill in the art. For example, the comparison of the level ofhyaluronan with a reference, control or predetermined level can be doneby an automated system, such as software program or intelligence systemthat is part of, or compatible with, the equipment (e.g. computerplatform) on which the assay is carried out. Alternatively, thiscomparison can be done by a physician or other trained or experiencedprofessional or technician.

E. TREATMENT OF SELECTED SUBJECTS WITH AN ANTI-HYALURONAN AGENT

The methods provided herein include methods of treating a tumor-bearingsubject with an anti-hyaluronan agent, for example ahyaluronan-degrading enzyme, where the subject has been selected fortreatment based on level of HA detected in the tumor. The methods oftreatment also include methods for assessing effects of treatment withan anti-hyaluronan agent, for example a hyaluronan-degrading enzyme,such as efficacy of treatment, such as for example, tumor inhibition orregression, or side effects of treatment, such as for example,musculoskeletal side effects. Combination therapies with one or moreadditional anti-cancer agents or one more agents to treat one or moreside effects of therapy with an anti-hyaluronan agent (e.g. ahyaluronan-degrading enzyme) also are provided.

1. Anti-Hyaluronan Agent

Anti-hyaluronan agents include agents that inhibit hyaluronan synthesisor degrade hyaluronan. Anti-hyaluronan agents, such as hyaluronandegrading enzymes, can be used to treat a hyaluronan-associated diseaseor condition, including tumors and cancers or inflammatory diseases orconditions. For example, HA accumulation, such as by altered hyaluronanmetabolism, distribution and function is associated with arthritis,immune and inflammatory disorders, pulmonary and vascular diseases andcancer (Morohashi et al. (2006) Biochem. Biophys. Res. Comm.,345:1454-1459). Such diseases can be treated by inhibiting HA synthesisor degrading HA (see e.g. Morohashi 2006; U.S. published application No.20100003238 and International published PCT Appl. No WO 2009/128917). Insome examples, such treatments that reduce hyaluronan levels on cellsand tissues can be associated with adverse side effects, such asmusculoskeletal side effects. Hence, treatment with ananti-hyaluronan-agent can further include treatment with acorticosteroid to ameliorate or reduce such side effects.

a. Agents that Inhibit Hyaluronan Synthesis

HA can be synthesized by three enzymes that are the products of threerelated mammalian genes identified as HA synthases, designated has-1,has-2 and has-3. Different cell types express different HAS enzymes andexpression of HAS mRNAs is correlated with HA biosynthesis. It is knownthat silencing HAS genes in tumor cells inhibits tumor growth andmetastasis. An anti-hyaluronan agent includes any agent that inhibits,reduces or downregulates the expression or level of an HA synthase. Suchagents are known to one of skill in the art or can be identified.

For example, downregulation of an HAS can be accomplished by providingoligonucleotides that specifically hybridize or otherwise interact withone or more nucleic acid molecules encoding an HAS. For example,anti-hyaluronan agents that inhibit hyaluronan synthesis includeantisense or sense molecules against an has gene. Such antisense orsense inhibition is typically based upon hydrogen bonding-basedhybridization of oligonucleotide strands or segments such that at leastone strand or segment is cleaved, degraded or otherwise renderedinoperable. In other examples, post-transcriptional gene silencing(PTGS), RNAi, ribozymes and DNAzymes can be employed. It is within thelevel of one skill in the art to generate such constructs based on thesequence of HAS1 (set forth in SEQ ID NO:219), HAS2 (set forth in SEQ IDNO:220) or HAS3 (set forth in SEQ ID NO:221). It is understood in theart that the sequence of an antisense or sense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. Moreover, an oligonucleotide can hybridize over one ormore segments such that intervening or adjacent segments are notinvolved in the hybridization event (e.g. a loop structure or hairpinstructure). Generally, the antisense or sense compounds have at least70% sequence complementarity to a target region within the targetnucleic acid, for example, 75% to 100% complementarity, such as 75%,80%, 85%, 90%, 95% or 100%. Exemplary sense or antisense molecules areknown in the art (see e.g. Chao et al. (2005) J. Biol. Chem.,280:27513-27522; Simpson et al. (2002) J. Biol. Chem., 277:10050-10057;Simpson et al. (2002) Am. J Path., 161:849; Nishida et al. (1999) J.Biol. Chem., 274:21893-21899; Edward et al. (2010) British JDermatology, 162:1224-1232; Udabage et al. (2005) Cancer Res., 65:6139;and published U.S. Patent application No. US20070286856).

Another exemplary anti-hyaluronan agent that is an HA synthesisinhibitor is 4-Methylumbelliferone (4-MU; 7-hydroxy-4-methylcoumarin) ora derivative thereof. 4-MU acts by reducing the UDP-GlcUA precursor poolthat is required for HA synthesis. For example, in mammalian cells, HAis synthesized by HAS using UDP-glucuronic acid (UGA) andUDP-N-acetyl-D-glucosamine precursors. 4-MU interferes with the processby which UGA is generated, thereby depleting the intracellular pool ofUGA and resulting in inhibition of HA synthesis. 4-MU is known to haveantitumor activity (see e.g. Lokeshwar et al. (2010) Cancer Res.,70:2613-23; Nakazawa et al. (2006) Cancer Chemother. Pharmacol.,57:165-170; Morohashi et al. (2006) Biochem. Biophys. Res. Comm.,345-1454-1459). Oral administration of 4-MU at 600 mg/kg/d reducesmetastases by 64% in the B16 melanoma model (Yoshihara et al. (2005)FEBS Lett., 579:2722-6). The structure of 4-MU is set forth below. Also,derivatives of 4-MU exhibit anti-cancer activity, in particular6,7-dihydrozy-4-methyl coumarin and 5,7-dihydroxy-4-methyl coumarin (seee.g. Morohashi et al. (2006) Biochem. Biophys. Res. Comm.,345-1454-1459).

Further exemplary anti-hyaluronan agents are tyrosine kinase inhibitors,such as Leflunomide (Arava), genistein or erbstatin. Leflunomide also isa pyrimidine synthesis inhibitor. Leflunomide is a known drug for thetreatment of Rheumatoid arthritis (RA), and also is effective intreating the rejection of allografts as well as xenografts. HA is knownto directly or indirectly contribute to RA (see e.g. Stuhlmeier (2005) JImmunol., 174:7376-7382). Tyrosine kinase inhibitors inhibit HAS1 geneexpression (Stuhlmeier 2005).

In one example, leflunomide, or derivatives thereof, generally areavailable as tablets containing 1-100 mg of active drug, for example, 1,5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg of drug. For thetreatment of a hyaluronan-associated disease and conditions, for exampleRheumatoid arthritis or a tumor or cancer, it is administered at 10 to500 mg per day, typically 100 mg per day. The dosage can be continued asneeded for treatment of the disease or condition, or can be tapered orreduced to successively lower doses. For example, for treatment ofRheumatoid arthristis, leflunomide can be administered at an initialloading dose of 100 mg per day for three days and then administered at acontinued dose of 20 mg/day.

b. Hyaluronan-Degrading Enzymes

Hyaluronan is an essential component of the extracellular matrix and amajor constituent of the interstitial barrier. By catalyzing thehydrolysis of hyaluronan, hyaluronan-degrading enzymes lower theviscosity of hyaluronan, thereby increasing tissue permeability andincreasing the absorption rate of fluids administered parenterally. Assuch, hyaluronan-degrading enzymes, such as hyaluronidases, have beenused, for example, as spreading or dispersing agents in conjunction withother agents, drugs and proteins to enhance their dispersion anddelivery.

Hyaluronan degrading enzymes act to degrade hyaluronan by cleavinghyaluronan polymers, which are composed of repeating disaccharidesunits, D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc),linked together via alternating β-1→4 and β-1→3 glycosidic bonds.Hyaluronan chains can reach about 25,000 disaccharide repeats or more inlength and polymers of hyaluronan can range in size from about 5,000 to20,000,000 Da in vivo. Accordingly, hyaluronan degrading enzymes for theuses and methods provided include any enzyme having the ability tocatalyze the cleavage of a hyaluronan disaccharide chain or polymer. Insome examples the hyaluronan degrading enzyme cleaves the β-1→4glycosidic bond in the hyaluronan chain or polymer. In other examples,the hyaluronan degrading enzyme catalyze the cleavage of the β-1→3glycosidic bond in the hyaluronan chain or polymer.

Hence, hyaluronan degrading enzymes, such as hyaluronidases, are afamily of enzymes that degrade hyaluronic acid, which is an essentialcomponent of the extracellular matrix and a major constituent of theinterstitial barrier. By catalyzing the hydrolysis of hyaluronic acid, amajor constituent of the interstitial barrier, hyaluronan degradingenzymes lower the viscosity of hyaluronic acid, thereby increasingtissue permeability. As such, hyaluronan degrading enzymes, such ashyaluronidases, have been used, for example, as a spreading ordispersing agent in conjunction with other agents, drugs and proteins toenhance their dispersion and delivery. Hyaluronan-degrading enzymes alsoare used as an adjuvant to increase the absorption and dispersion ofother injected drugs, for hypodermoclysis (subcutaneous fluidadministration), and as an adjunct in subcutaneous urography forimproving resorption of radiopaque agents. Hyaluronan-degrading enzymes,for example, hyaluronidase can be used in applications of ophthalmicprocedures, for example, peribulbar and sub-Tenon's block in localanesthesia prior to ophthalmic surgery. Hyaluronidase also can be usedin other therapeutic and cosmetic uses, for example, by promotingakinesia in cosmetic surgery, such as blepharoplasties and face lifts.

Various forms of hyaluronan degrading enzymes, including hyaluronidaseshave been prepared and approved for therapeutic use in subjects,including humans. The provided compositions and methods can be used, viathese and other therapeutic uses, to treat hyaluronan-associateddiseases and conditions. For example, animal-derived hyaluronidasepreparations include Vitrase (ISTA Pharmaceuticals), a purified ovinetesticular hyaluronidase, Amphadase (Amphastar Pharmaceuticals), abovine testicular hyaluronidase and Hydase (Prima Pharm Inc.), a bovinetesticular hyaluronidase. It is understood that any animal-derivedhyaluronidase preparation can be used in the methods and uses providedherein (see, e.g., U.S. Pat. Nos. 2,488,564, 2,488,565, 2,676,139,2,795,529, 2,806,815, 2,808,362, 5,747,027 and 5,827,721 and InternationPCT Application No. WO2005/118799). Hylenex (Halozyme Therapeutics) is ahuman recombinant hyaluronidase produced by genetically engineeredChinese Hamster Ovary (CHO) cells containing nucleic acid encodingsoluble forms of PH20, designated rHuPH20.

Exemplary of hyaluronan degrading enzymes in the compositions andmethods provided herein are soluble hyaluronidases. Other exemplaryhyaluronan degrading enzymes include, but are not limited to particularchondroitinases and lyases that have the ability to cleave hyaluronan.

As described below, hyaluronan-degrading enzymes exist in membrane-boundor soluble forms that are secreted from cells. For purposes herein,soluble hyaluronan-degrading enzymes are provided for use in themethods, uses, compositions or combinations herein. Thus, wherehyaluronan-degrading enzymes include a glycosylphosphatidylinositol(GPI) anchor and/or are otherwise membrane-anchored or insoluble, suchhyaluronan-degrading enzymes are provided herein in soluble form bytruncation or deletion of the GPI anchor to render the enzyme secretedand soluble. Thus, hyaluronan-degrading enzymes include truncatedvariants, e.g. truncated to remove all or a portion of a GPI anchor.Hyaluronan-degrading enzymes provide herein also include allelic orspecies variants or other variants, of a soluble hyaluronan-degradingenzyme. For example, hyaluronan degrading enzymes can contain one ormore variations in its primary sequence, such as amino acidsubstitutions, additions and/or deletions. A variant of ahyaluronan-degrading enzyme generally exhibits at least or about 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity compared to the hyaluronan-degrading enzyme notcontaining the variation. Any variation can be included in thehyaluronan degrading enzyme for the purposes herein provided the enzymeretains hyaluronidase activity, such as at least or about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or more of the activity of a hyaluronan degrading enzyme notcontaining the variation (as measured by in vitro and/or in vivo assayswell known in the art and described herein).

Where the methods and uses provided herein describe the use of a solublehyaluronidase, accordingly any hyaluronan degrading enzyme, generally asoluble hyaluronan degrading enzyme, can be used. It is understood thatany hyaluronidase can be used in the methods and uses provided herein(see, e.g., U.S. Pat. No. 7,767,429 and U.S. Publication Nos.US20040268425 and US20100143457).

i. Hyaluronidases

Hyaluronidases are members of a large family of hyaluronan degradingenzymes. There are three general classes of hyaluronidases:mammalian-type hyaluronidases, bacterial hyaluronidases andhyaluronidases from leeches, other parasites and crustaceans. Suchenzymes can be used in the compositions, combinations and methodsprovided herein.

(1) Mammalian-Type Hyaluronidases

Mammalian-type hyaluronidases (EC 3.2.1.35) areendo-β-N-acetyl-hexosaminidases that hydrolyze the β-1→4 glycosidic bondof hyaluronan into various oligosaccharide lengths such astetrasaccharides and hexasaccharides. These enzymes have both hydrolyticand transglycosidase activities, and can degrade hyaluronan andchondroitin sulfates (CS), generally C4-S and C6-S. Hyaluronidases ofthis type include, but are not limited to, hyaluronidases from cows(bovine) (SEQ ID NOS:10, 11 and 64 and BH55 (U.S. Pat. Nos. 5,747,027and 5,827,721), nucleic acid molecules set forth in SEQ ID NOS:190-192),sheep (Ovis aries) (SEQ ID NO: 26, 27, 63 and 65, nucleic acid moleculesset forth in SEQ ID NOS:66 and 193-194), yellow jacket wasp (SEQ IDNOS:12 and 13), honey bee (SEQ ID NO:14), white-face hornet (SEQ IDNO:15), paper wasp (SEQ ID NO:16), mouse (SEQ ID NOS:17-19, 32), pig(SEQ ID NOS:20-21), rat (SEQ ID NOS:22-24, 31), rabbit (SEQ ID NO:25),orangutan (SEQ ID NO:28), cynomolgus monkey (SEQ ID NO:29), guinea pig(SEQ ID NO:30), chimpanzee (SEQ ID NO:101), rhesus monkey (SEQ IDNO:102), and human hyaluronidases (SEQ ID NOS:1-2, 36-39). Exemplary ofhyaluronidases in the compositions, combinations and methods providedherein are soluble hyaluronidases.

Mammalian hyaluronidases can be further subdivided into those that areneutral active, predominantly found in testes extracts, and acid active,predominantly found in organs such as the liver. Exemplary neutralactive hyaluronidases include PH20, including but not limited to, PH20derived from different species such as ovine (SEQ ID NOS:27, 63 and 65),bovine (SEQ ID NO:11 and 64) and human (SEQ ID NO:1). Human PH20 (alsoknown as SPAM1 or sperm surface protein PH20), is generally attached tothe plasma membrane via a glycosylphosphatidyl inositol (GPI) anchor. Itis naturally involved in sperm-egg adhesion and aids penetration bysperm of the layer of cumulus cells by digesting hyaluronic acid.

Besides human PH20 (also termed SPAM1), five hyaluronidase-like geneshave been identified in the human genome, HYAL1, HYAL2, HYAL3, HYAL4 andHYALP1. HYALP1 is a pseudogene, and HYAL3 (SEQ ID NO:38) has not beenshown to possess enzyme activity toward any known substrates. HYAL4(precursor polypeptide set forth in SEQ ID NO:39) is a chondroitinaseand exhibits little activity towards hyaluronan. HYAL1 (precursorpolypeptide set forth in SEQ ID NO:36) is the prototypical acid-activeenzyme and PH20 (precursor polypeptide set forth in SEQ ID NO:1) is theprototypical neutral-active enzyme. Acid-active hyaluronidases, such asHYAL1 and HYAL2 (precursor polypeptide set forth in SEQ ID NO:37)generally lack catalytic activity at neutral pH (i.e. pH 7). Forexample, HYAL1 has little catalytic activity in vitro over pH 4.5 (Frostet al. (1997) Anal. Biochem. 251:263-269). HYAL2 is an acid-activeenzyme with a very low specific activity in vitro. Thehyaluronidase-like enzymes also can be characterized by those which aregenerally attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor such as human HYAL2 and human PH20(Danilkovitch-Miagkova et al. (2003) Proc Natl Acad Sci USA100(8):4580-5), and those which are generally soluble such as humanHYAL1 (Frost et al. (1997) Biochem Biophys Res Commun. 236(1):10-5).

(a) PH20

PH20, like other mammalian hyaluronidases, is anendo-β-N-acetyl-hexosaminidase that hydrolyzes the β1→4 glycosidic bondof hyaluronic acid into various oligosaccharide lengths such astetrasaccharides and hexasaccharides. It has both hydrolytic andtransglycosidase activities and can degrade hyaluronic acid andchondroitin sulfates, such as C4-S and C6-S. PH20 is naturally involvedin sperm-egg adhesion and aids penetration by sperm of the layer ofcumulus cells by digesting hyaluronic acid. PH20 is located on the spermsurface, and in the lysosome-derived acrosome, where it is bound to theinner acrosomal membrane. Plasma membrane PH20 has hyaluronidaseactivity only at neutral pH, while inner acrosomal membrane PH20 hasactivity at both neutral and acid pH. In addition to being ahyaluronidase, PH20 is reported to be a receptor for HA-induced cellsignaling, and a receptor for the zona pellucida surrounding the oocyte.

Exemplary PH20 proteins include, but are not limited to, human(precursor polypeptide set forth in SEQ ID NO:1, mature polypeptide setforth in SEQ ID NO: 2), chimpanzee (SEQ ID NO:101), Rhesus monkey (SEQID NO:102) bovine (SEQ ID NOS: 11 and 64), rabbit (SEQ ID NO: 25), ovinePH20 (SEQ ID NOS: 27, 63 and 65), Cynomolgus monkey (SEQ ID NO: 29),guinea pig (SEQ ID NO: 30), rat (SEQ ID NO: 31) and mouse (SEQ ID NO:32) PH20 polypeptides.

Bovine PH20 is a 553 amino acid precursor polypeptide (SEQ ID NO:11).Alignment of bovine PH20 with the human PH20 shows only weak homology,with multiple gaps existing from amino acid 470 through to therespective carboxy termini due to the absence of a GPI anchor in thebovine polypeptide (see e.g., Frost GI (2007) Expert Opin. Drug. Deliv.4: 427-440). In fact, clear GPI anchors are not predicted in many otherPH20 species besides humans. Thus, PH20 polypeptides produced from ovineand bovine naturally exist as soluble forms. Though bovine PH20 existsvery loosely attached to the plasma membrane, it is not anchored via aphospholipase sensitive anchor (Lalancette et al. (2001) Biol Reprod.65(2):628-36). This unique feature of bovine hyaluronidase has permittedthe use of the soluble bovine testes hyaluronidase enzyme as an extractfor clinical use (Wydase®, Hyalase®).

The human PH20 mRNA transcript is normally translated to generate a 509amino acid precursor polypeptide (SEQ ID NO:1) containing a 35 aminoacid signal sequence at the N-terminus (amino acid residue positions1-35) and a 19 amino acid glycosylphosphatidylinositol (GPI) anchorattachment signal sequence at the C-terminus (amino acid residuepositions 491-509). The mature PH20 therefore, is a 474 amino acidpolypeptide set forth in SEQ ID NO:2. Following transport of theprecursor polypeptide to the ER and removal of the signal peptide, theC-terminal GPI-attachment signal peptide is cleaved to facilitatecovalent attachment of a GPI anchor to the newly-formed C-terminal aminoacid at the amino acid position corresponding to position 490 of theprecursor polypeptide set forth in SEQ ID NO:1. Thus, a 474 amino acidGPI-anchored mature polypeptide with an amino acid sequence set forth inSEQ ID NO:2 is produced.

Human PH20 exhibits hyaluronidase activity at neutral and acid pH. Inone aspect, human PH20 is the prototypical neutral-active hyaluronidasethat is generally locked to the plasma membrane via a GPI anchor. Inanother aspect, PH20 is expressed on the inner acrosomal membrane whereit has hyaluronidase activity at neutral and acid pH. PH20 contains twocatalytic sites at distinct regions of the polypeptide: the Peptide 1and Peptide 3 regions (Cherr et al. (2001) Matrix Biology 20:515-525).Evidence indicates that the Peptide 1 region of PH20, which correspondsto amino acid positions 107-137 of the mature polypeptide set forth inSEQ ID NO:2 and positions 142-172 of the precursor polypeptide set forthin SEQ ID NO:1, is required for enzyme activity at neutral pH. Aminoacids at positions 111 and 113 (corresponding to the mature PH20polypeptide set forth in SEQ ID NO:2) within this region are reported tobe important for activity, as mutagenesis by amino acid replacementresults in PH20 polypeptides with 3% hyaluronidase activity orundetectable hyaluronidase activity, respectively, compared to thewild-type PH20 (Arming et al., (1997) Eur. J. Biochem. 247:810-814).

The Peptide 3 region, which corresponds to amino acid positions 242-262of the mature polypeptide set forth in SEQ ID NO:2, and positions277-297 of the precursor polypeptide set forth in SEQ ID NO: 1, isreported to be important for enzyme activity at acidic pH. Within thisregion, amino acids at positions 249 and 252 of the mature PH20polypeptide are reported to be essential for activity as mutagenesis ofeither results in a polypeptide essentially devoid of activity (Arminget al., (1997) Eur. J. Biochem. 247:810-814).

In addition to the catalytic sites, PH20 also contains ahyaluronan-binding site. Experimental evidence indicate that this siteis located in the Peptide 2 region, which corresponds to amino acidpositions 205-235 of the precursor polypeptide set forth in SEQ ID NO: 1and positions 170-200 of the mature polypeptide set forth in SEQ IDNO:2. This region is highly conserved among hyaluronidases and issimilar to the heparin binding motif. Mutation of the arginine residueat position 176 (corresponding to the mature PH20 polypeptide set forthin SEQ ID NO:2) to a glycine results in a polypeptide with only about 1%of the hyaluronidase activity of the wild type polypeptide (Arming etal., (1997) Eur. J. Biochem. 247:810-814).

There are seven potential glycosylation sites, including N-linkedglycosylation sites, in human PH20 at N82, N166, N235, N254, N368, N393,5490 of the polypeptide exemplified in SEQ ID NO: 1. Because amino acids36 to 464 of SEQ ID NO:1 is reported to contain the minimally activehuman PH20 hyaluronidase domain, the glycosylation site S490 is notrequired for proper hyaluronidase activity. There are six disulfidebonds in human PH20. Two disulfide bonds between the cysteine residuesC60 and C351 and between C224 and C238 of the polypeptide exemplified inSEQ ID NO: 1 (corresponding to residues C25 and C316, and C189 and C203of the mature polypeptide set forth in SEQ ID NO:2, respectively). Afurther four disulfide bonds are formed between the cysteine residuesC376 and C387; between C381 and C435; between C437 and C443; and betweenC458 and C464 of the polypeptide exemplified in SEQ ID NO: 1(corresponding to residues C341 and C352; between C346 and C400; betweenC402 and C408; and between C423 and C429 of the mature polypeptide setforth in SEQ ID NO:2, respectively).

(2) Other Hyaluronidases

Bacterial hyaluronidases (EC 4.2.2.1 or EC 4.2.99.1) degrade hyaluronanand, to various extents, chondroitin sulfates and dermatan sulfates.Hyaluronan lyases isolated from bacteria differ from hyaluronidases(from other sources, e.g., hyaluronoglucosaminidases, EC 3.2.1.35) bytheir mode of action. They are endo-β-N-acetylhexosaminidases thatcatalyze an elimination reaction, rather than hydrolysis, of theβ1→4-glycosidic linkage between N-acetyl-beta-D-glucosamine andD-glucuronic acid residues in hyaluronan, yielding3-(4-deoxy-β-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine tetra- andhexasaccharides, and disaccharide end products. The reaction results inthe formation of oligosaccharides with unsaturated hexuronic acidresidues at their nonreducing ends.

Exemplary hyaluronidases from bacteria for use in the compositions,combinations and methods provided include, but are not limited to,hyaluronan degrading enzymes in microorganisms, including strains ofArthrobacter, Bdellovibrio, Clostridium, Micrococcus, Streptococcus,Peptococcus, Propionibacterium, Bacteroides, and Streptomyces.Particular examples of such strains and enzymes include, but are notlimited to Arthrobacter sp. (strain FB24 (SEQ ID NO:67)), Bdellovibriobacteriovorus (SEQ ID NO:68), Propionibacterium acnes (SEQ ID NO:69),Streptococcus agalactiae ((SEQ ID NO:70); 18RS21 (SEQ ID NO:71);serotype Ia (SEQ ID NO:72); serotype III (SEQ ID NO:73)), Staphylococcusaureus (strain COL (SEQ ID NO:74); strain MRSA252 (SEQ ID NOS:75 and76); strain MSSA476 (SEQ ID NO:77); strain NCTC 8325 (SEQ ID NO:78);strain bovine RF122 (SEQ ID NOS:79 and 80); strain USA300 (SEQ IDNO:81)), Streptococcus pneumoniae ((SEQ ID NO:82); strain ATCCBAA-255/R6 (SEQ ID NO:83); serotype 2, strain D39/NCTC 7466 (SEQ IDNO:84)), Streptococcus pyogenes (serotype M1 (SEQ ID NO:85); serotypeM2, strain MGAS 10270 (SEQ ID NO:86); serotype M4, strain MGAS 10750(SEQ ID NO:87); serotype M6 (SEQ ID NO:88); serotype M12, strainMGAS2096 (SEQ ID NOS:89 and 90); serotype M12, strain MGAS9429 (SEQ IDNO:91); serotype M28 (SEQ ID NO:92)), Streptococcus suis (SEQ IDNOS:93-95); Vibrio fischeri (strain ATCC 700601/ES114 (SEQ ID NO:96)),and the Streptomyces hyaluronolyticus hyaluronidase enzyme, which isspecific for hyaluronic acid and does not cleave chondroitin orchondroitin sulfate (Ohya, T. and Kaneko, Y. (1970) Biochim. Biophys.Acta 198:607). Hyaluronidases from leeches, other parasites, andcrustaceans (EC 3.2.1.36) are endo-β-glucuronidases that generate tetra-and hexasaccharide end-products. These enzymes catalyze hydrolysis of1→3-linkages between β-D-glucuronate and N-acetyl-D-glucosamine residuesin hyaluronate. Exemplary hyaluronidases from leeches include, but arenot limited to, hyaluronidase from Hirudinidae (e.g., Hirudomedicinalis), Erpobdellidae (e.g., Nephelopsis obscura and Erpobdellapunctata), Glossiphoniidae (e.g., Desserobdella picta, Helobdellastagnalis, Glossiphonia complanata, Placobdella ornata and Theromyzonsp.) and Haemopidae (Haemopis marmorata) (Hovingh et al. (1999) CompBiochem Physiol B Biochem Mol Biol. 124(3):319-26). An exemplaryhyaluronidase from bacteria that has the same mechanism of action as theleech hyaluronidase is that from the cyanobacteria, Synechococcus sp.(strain RCC307, SEQ ID NO:97).

(3) Other Hyaluronan Degrading Enzymes

In addition to the hyaluronidase family, other hyaluronan degradingenzymes can be used in the compositions, combinations and methodsprovided. For example, enzymes, including particular chondroitinases andlyases, that have the ability to cleave hyaluronan can be employed.Exemplary chondroitinases that can degrade hyaluronan include, but arenot limited to, chondroitin ABC lyase (also known as chondroitinaseABC), chondroitin AC lyase (also known as chondroitin sulfate lyase orchondroitin sulfate eliminase) and chondroitin C lyase. Methods forproduction and purification of such enzymes for use in the compositions,combinations, and methods provided are known in the art (e.g., U.S. Pat.No. 6,054,569; Yamagata, et al. (1968) J. Biol. Chem. 243(7):1523-1535;Yang et al. (1985) J. Biol. Chem. 160(30):1849-1857).

Chondroitin ABC lyase contains two enzymes, chondroitin-sulfate-ABCendolyase (EC 4.2.2.20) and chondroitin-sulfate-ABC exolyase (EC4.2.2.21) (Hamai et al. (1997) J Biol Chem. 272(14):9123-30), whichdegrade a variety of glycosaminoglycans of the chondroitin-sulfate- anddermatan-sulfate type. Chondroitin sulfate, chondroitin-sulfateproteoglycan and dermatan sulfate are the preferred substrates forchondroitin-sulfate-ABC endolyase, but the enzyme also can act onhyaluronan at a lower rate. Chondroitin-sulfate-ABC endolyase degrades avariety of glycosaminoglycans of the chondroitin-sulfate- anddermatan-sulfate type, producing a mixture of Δ4-unsaturatedoligosaccharides of different sizes that are ultimately degraded toΔ4-unsaturated tetra- and disaccharides. Chondroitin-sulfate-ABCexolyase has the same substrate specificity but removes disaccharideresidues from the non-reducing ends of both polymeric chondroitinsulfates and their oligosaccharide fragments produced bychondroitin-sulfate-ABC endolyase (Hamai, A. et al. (1997) J. Biol.Chem. 272:9123-9130). Exemplary chondroitin-sulfate-ABC endolyases andchondroitin-sulfate-ABC exolyases include, but are not limited to, thosefrom Proteus vulgaris and Pedobacter heparinus (the Proteus vulgarischondroitin-sulfate-ABC endolyase is set forth in SEQ ID NO: 98 (Sato etal. (1994) Appl. Microbiol. Biotechnol. 41(1):39-46).

Chondroitin AC lyase (EC 4.2.2.5) is active on chondroitin sulfates Aand C, chondroitin and hyaluronic acid, but is not active on dermatansulfate (chondroitin sulfate B). Exemplary chondroitinase AC enzymesfrom the bacteria include, but are not limited to, those from Pedobacterheparinus and Victivallis vadensis, set forth in SEQ ID NOS:99 and 100,respectively, and Arthrobacter aurescens (Tkalec et al. (2000) Appliedand Environmental Microbiology 66(1):29-35; Ernst et al. (1995) CriticalReviews in Biochemistry and Molecular Biology 30(5):387-444).

Chondroitinase C cleaves chondroitin sulfate C producing tetrasaccharideplus an unsaturated 6-sulfated disaccharide (delta Di-6S). It alsocleaves hyaluronic acid producing unsaturated non-sulfated disaccharide(delta Di-OS). Exemplary chondroitinase C enzymes from the bacteriainclude, but are not limited to, those from Streptococcus andFlavobacterium (Hibi et al. (1989) FEMS-Microbiol-Lett. 48(2):121-4;Michelacci et al. (1976) J. Biol. Chem. 251:1154-8; Tsuda et al. (1999)Eur. J. Biochem. 262:127-133).

ii. Soluble Hyaluronan Degrading Enzymes

Provided in the compositions, combinations, uses and methods herein aresoluble hyaluronan degrading enzymes, including soluble hyaluronidases.Soluble hyaluronan degrading enzymes include any hyaluronan degradingenzymes that are secreted from cells (e.g. CHO cell) upon expression andexist in soluble form. Such enzymes include, but are not limited to,soluble hyaluronidases, including non-human soluble hyaluronidases,including non-human animal soluble hyaluronidases, bacterial solublehyaluronidases and human hyaluronidases, Hyal1, bovine PH20 and ovinePH20, allelic variants thereof and other variants thereof. For example,included among soluble hyaluronan degrading enzymes are any hyaluronandegrading enzymes that have been modified to be soluble. For example,hyaluronan degrading enzymes that contain a GPI anchor can be madesoluble by truncation of and removal of all or a portion of the GPIanchor. In one example, the human hyaluronidase PH20, which is normallymembrane anchored via a GPI anchor, can be made soluble by truncation ofand removal of all or a portion of the GPI anchor at the C-terminus.

Soluble hyaluronan degrading enzymes also include neutral active andacid active hyaluronidases. Depending on factors, such as, but notlimited to, the desired level of activity of the enzyme followingadministration and/or site of administration, neutral active and acidactive hyaluronidases can be selected. In a particular example, thehyaluronan degrading enzyme for use in the compositions, combinationsand methods herein is a soluble neutral active hyaluronidase.

Exemplary of a soluble hyaluronidase is PH20 from any species, such asany set forth in any of SEQ ID NOS: 1, 2, 11, 25, 27, 29-32, 63-65 and101-102, or truncated forms thereof lacking all or a portion of theC-terminal GPI anchor, so long as the hyaluronidase is soluble (secretedupon expression) and retains hyaluronidase activity. Also included amongsoluble hyaluronidases are allelic variants or other variants of any ofSEQ ID NOS:1, 2, 11, 25, 27, 29-32, 63-65 and 101-102, or truncatedforms thereof. Allelic variants and other variants are known to one ofskill in the art, and include polypeptides having 60%, 70%, 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%., 97%, 98%, 99% or more sequence identityto any of SEQ ID NOS: 1, 2, 11, 25, 27, 29-32, 63-65 and 101-102, ortruncated forms thereof. Amino acid variants include conservative andnon-conservative mutations. It is understood that residues that areimportant or otherwise required for the activity of a hyaluronidase,such as any described above or known to skill in the art, are generallyinvariant and cannot be changed. These include, for example, active siteresidues. Thus, for example, amino acid residues 111, 113 and 176(corresponding to residues in the mature PH20 polypeptide set forth inSEQ ID NO:2) of a human PH20 polypeptide, or soluble form thereof, aregenerally invariant and are not altered. Other residues that conferglycosylation and formation of disulfide bonds required for properfolding also can be invariant.

In some instances, the soluble hyaluronan degrading enzyme is normallyGPI-anchored (such as, for example, human PH20) and is rendered solubleby truncation at the C-terminus. Such truncation can remove all of theGPI anchor attachment signal sequence, or can remove only some of theGPI anchor attachment signal sequence. The resulting polypeptide,however, is soluble. In instances where the soluble hyaluronan degradingenzyme retains a portion of the GPI anchor attachment signal sequence,1, 2, 3, 4, 5, 6, 7 or more amino acid residues in the GPI-anchorattachment signal sequence can be retained, provided the polypeptide issoluble. Polypeptides containing one or more amino acids of the GPIanchor are termed extended soluble hyaluronan degrading enzymes. One ofskill in the art can determine whether a polypeptide is GPI-anchoredusing methods well known in the art. Such methods include, but are notlimited to, using known algorithms to predict the presence and locationof the GPI-anchor attachment signal sequence and w-site, and performingsolubility analyses before and after digestion withphosphatidylinositol-specific phospholipase C (PI-PLC) or D (PI-PLD).

Extended soluble hyaluronan degrading enzymes can be produced by makingC-terminal truncations to any naturally GPI-anchored hyaluronandegrading enzyme such that the resulting polypeptide is soluble andcontains one or more amino acid residues from the GPI-anchor attachmentsignal sequence (see, e.g., U.S. Published Pat. Appl. No.US20100143457). Exemplary extended soluble hyaluronan degrading enzymesthat are C-terminally truncated but retain a portion of the GPI anchorattachment signal sequence include, but are not limited to, extendedsoluble PH20 (esPH20) polypeptides of primate origin, such as, forexample, human and chimpanzee esPH20 polypeptides. For example, theesPH20 polypeptides can be made by C-terminal truncation of any of themature or precursor polypeptides set forth in SEQ ID NOS:1, 2 or 101, orallelic or other variation thereof, including active fragment thereof,wherein the resulting polypeptide is soluble and retains one or moreamino acid residues from the GPI-anchor attachment signal sequence.Allelic variants and other variants are known to one of skill in theart, and include polypeptides having 60%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95% or more sequence identity to any of SEQ ID NOS: 1 or 2. TheesPH20 polypeptides provided herein can be C-terminally truncated by 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids compared to the wild typepolypeptide, such as a polypeptide with a sequence set forth in SEQ IDNOS: 1, 2 or 101, provided the resulting esPH20 polypeptide is solubleand retains 1 or more amino acid residues from the GPI-anchor attachmentsignal sequence.

Typically, for use in the compositions, combinations and methods herein,a soluble human hylauronan degrading enzyme, such as a soluble humanPH20, is used. Although hylauronan degrading enzymes, such as PH20, fromother animals can be utilized, such preparations are potentiallyimmunogenic, since they are animal proteins. For example, a significantproportion of patients demonstrate prior sensitization secondary toingested foods, and since these are animal proteins, all patients have arisk of subsequent sensitization. Thus, non-human preparations may notbe suitable for chronic use. If non-human preparations are desired, itis contemplated herein that such polypeptides can be prepared to havereduced immunogenicity. Such modifications are within the level of oneof skill in the art and can include, for example, removal and/orreplacement of one or more antigenic epitopes on the molecule.

Hyaluronan degrading enzymes, including hyaluronidases (e.g., PH20),used in the methods herein can be recombinantly produced or can bepurified or partially-purified from natural sources, such as, forexample, from testes extracts. Methods for production of recombinantproteins, including recombinant hyaluronan degrading enzymes, areprovided elsewhere herein and are well known in the art.

(1) Soluble Human PH20

Exemplary of a soluble hyaluronidase is soluble human PH20. Solubleforms of recombinant human PH20 have been produced and can be used inthe compositions, combinations and methods described herein. Theproduction of such soluble forms of PH20 is described in U.S. PublishedPatent Application Nos. US20040268425; US20050260186, US20060104968,US20100143457 and International PCT application No. WO2009111066. Forexample, soluble PH20 polypeptides, include C-terminally truncatedvariant polypeptides that include a sequence of amino acids in SEQ IDNO:1, or have at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98% sequenceidentity to a sequence of amino acids included in SEQ ID NO:1, retainhyaluronidase activity and are soluble. Included among thesepolypeptides are soluble PH20 polypeptides that completely lack all or aportion of the GPI-anchor attachment signal sequence.

Also included are extended soluble PH20 (esPH20) polypeptides thatcontain at least one amino acid of the GPI anchor. Thus, instead ofhaving a GPI-anchor covalently attached to the C-terminus of the proteinin the ER and being anchored to the extracellular leaflet of the plasmamembrane, these polypeptides are secreted and are soluble. C-terminallytruncated PH20 polypeptides can be C-terminally truncated by 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60 or more amino acids compared to the full length wildtype polypeptide, such as a full length wild type polypeptide with asequence set forth in SEQ ID NOS:1 or 2, or allelic or species variantsor other variants thereof.

For example, soluble forms include, but are not limited to, C-terminaltruncated polypeptides of human PH20 set forth in SEQ ID NO:1 having aC-terminal amino acid residue 467, 468, 469, 470, 471, 472, 473, 474,475, 476, 477, 478, 479, 480, 481, 482 and 483, 484, 485, 486, 487, 488,489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 of thesequence of amino acids set forth in SEQ ID NO:1, or polypeptides thatexhibit at least 85% identity thereto. Soluble forms of human PH20generally include those that contain amino acids 36-464 set forth in SEQID NO:1. For example, when expressed in mammalian cells, the 35 aminoacid N-terminal signal sequence is cleaved during processing, and themature form of the protein is secreted. Thus, the mature solublepolypeptides contain amino acids 36 to 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482 and 483 of SEQ ID NO:1.Table 3 provides non-limiting examples of exemplary C-terminallytruncated PH20 polypeptides, including C-terminally truncated solublePH20 polypeptides. In Table 3 below, the length (in amino acids) of theprecursor and mature polypeptides, and the sequence identifier (SEQ IDNO) in which exemplary amino acid sequences of the precursor and maturepolypeptides of the C-terminally truncated PH20 proteins are set forth,are provided. The wild-type PH20 polypeptide also is included in Table 3for comparison. In particular, exemplary of soluble hyaluronidases aresoluble human PH20 polypeptides that are 442, 443, 444, 445, 446 or 447amino acids in length, such as set forth in any of SEQ ID NOS: 4-9, orallelic or species variants or other variants thereof.

TABLE 3 Exemplary C-terminally truncated PH20 polypeptides PrecursorPrecursor Mature Mature (amino SEQ (amino SEQ Polypeptide acids) ID NOacids) ID NO wildtype 509 1 474 2 SPAM1-SILF 500 139 465 183 SPAM-VSIL499 106 464 150 SPAM1-IVSI 498 140 463 184 SPAM1-FIVS 497 107 462 151SPAM1-MFIV 496 141 461 185 SPAM1-TMFI 495 108 460 152 SPAM1-ATMF 494 142459 186 SPAM1-SATM 493 109 458 153 SPAM1-LSAT 492 143 457 187 SPAM1-TLSA491 110 456 154 SPAM1-STLS 490 112 455 156 SPAM1-PSTL 489 111 454 155SPAM1-SPST 488 144 453 188 SPAM1-ASPS 487 113 452 157 SPAM1-NASP 486 145451 189 SPAM1-YNAS 485 114 450 158 SPAM1-FYNA 484 115 449 159 SPAM1-IFYN483 46 448 48 SPAM1-QIFY 482 3 447 4 SPAM1-PQIF 481 45 446 5 SPAM1-EPQI480 44 445 6 SPAM1-EEPQ 479 43 444 7 SPAM1-TEEP 478 42 443 8 SPAM1-ETEE477 41 442 9 SPAM1-METE 476 116 441 160 SPAM1-PMET 475 117 440 161SPAM1-PPME 474 118 439 162 SPAM1-KPPM 473 119 438 163 SPAM1-LKPP 472 120437 164 SPAM1-FLKP 471 121 436 165 SPAM1-AFLK 470 122 435 166 SPAM1-DAFL469 123 434 167 SPAM1-IDAF 468 124 433 168 SPAM1-CIDA 467 40 432 47SPAM1-VCID 466 125 431 169 SPAM1-GVCI 465 126 430 170For example, exemplary C-terminally truncated PH20 polypeptides thatexhibit hyaluronidase activity, are secreted from cells and are solubleinclude any of the mature forms of a truncated human PH20 set forth inTable 3, or variants thereof that exhibit hyaluronidase activity. Forexample, the PH20 or truncated form thereof contains the sequence ofamino acids set forth in any of SEQ ID NOS: 4-9, 47, 48, 150-170 and183-189 or a sequence of amino acids that exhibits at least 85% sequenceidentity to any of SEQ ID NOS: 4-9, 47, 48, 150-170 and 183-189. Forexample, the PH20 polypeptide can exhibit at least 85%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to any of SEQ ID NOS: 4-9, 47, 48, 150-170 and 183-189.

Generally soluble forms of PH20 are produced using protein expressionsystems that facilitate correct N-glycosylation to ensure thepolypeptide retains activity, since glycosylation is important for thecatalytic activity and stability of hyaluronidases. Such cells include,for example Chinese Hamster Ovary (CHO) cells (e.g. DG44 CHO cells).

(2) rHuPH20

Recombinant soluble forms of human PH20 have been generated and can beused in the compositions, combinations and methods provided herein. Thegeneration of such soluble forms of recombinant human PH20 aredescribed, for example, in U.S. Published Patent Application Nos.US20040268425; US 20050260186; US20060104968; US20100143457; andInternational PCT Appl. No. WO2009111066. Exemplary of such polypeptidesare those generated by expression of a nucleic acid molecule encodingamino acids 1-482 (set forth in SEQ ID NO:3). Such an exemplary nucleicacid molecule is set forth in SEQ ID NO:49. Post translationalprocessing removes the 35 amino acid signal sequence, leaving a 447amino acid soluble recombinant human PH20 (SEQ ID NO:4). As produced inthe culture medium there is heterogeneity at the C-terminus such thatthe product, designated rHuPH20, includes a mixture of species that caninclude any one or more of SEQ ID NOS. 4-9 in various abundance.Typically, rHuPH20 is produced in cells that facilitate correctN-glycosylation to retain activity, such as CHO cells (e.g. DG44 CHOcells).

iii. Glycosylation of Hyaluronan Degrading Enzymes

Glycosylation, including N- and O-linked glycosylation, of somehyaluronan degrading enzymes, including hyaluronidases, can be importantfor their catalytic activity and stability. While altering the type ofglycan modifying a glycoprotein can have dramatic affects on a protein'santigenicity, structural folding, solubility, and stability, mostenzymes are not thought to require glycosylation for optimal enzymeactivity. For some hyaluronidases, removal of N-linked glycosylation canresult in near complete inactivation of the hyaluronidase activity.Thus, for such hyaluronidases, the presence of N-linked glycans iscritical for generating an active enzyme.

N-linked oligosaccharides fall into several major types (oligomannose,complex, hybrid, sulfated), all of which have (Man) 3-GlcNAc-GlcNAc-cores attached via the amide nitrogen of Asn residues that fall within-Asn-Xaa-Thr/Ser- sequences (where Xaa is not Pro). Glycosylation at an-Asn-Xaa-Cys-site has been reported for coagulation protein C. In someinstances, a hyaluronan degrading enzyme, such as a hyaluronidase, cancontain both N-glycosidic and 0-glycosidic linkages. For example, PH20has O-linked oligosaccharides as well as N-linked oligosaccharides.There are seven potential glycosylation sites at N82, N166, N235, N254,N368, N393, 5490 of human PH20 exemplified in SEQ ID NO: 1. Amino acidresidues N82, N166 and N254 are occupied by complex type glycans whereasamino acid residues N368 and N393 are occupied by high mannose typeglycans. Amino acid residue N235 is occupied by approximately 80% highmannose type glycans and 20% complex type glycans. As noted above,O-linked glycosylation at 5490 is not required for hyaluronidaseactivity.

In some examples, the hyaluronan degrading enzymes for use in thecompositions, combinations and/or methods provided are glycosylated atone or all of the glycosylation sites. For example, for human PH20, or asoluble form thereof, 2, 3, 4, 5, or 6 of the N-glycosylation sitescorresponding to amino acids N82, N166, N235, N254, N368, and N393 ofSEQ ID NO: 1 are glycosylated. In some examples the hyaluronan degradingenzymes are glycosylated at one or more native glycosylation sites. Inother examples, the hyaluronan degrading enzymes are modified at one ormore non-native glycosylation sites to confer glycosylation of thepolypeptide at one or more additional site. In such examples, attachmentof additional sugar moieties can enhance the pharmacokinetic propertiesof the molecule, such as improved half-life and/or improved activity.

In other examples, the hyaluronan degrading enzymes for use in thecompositions, combinations and/or methods provided herein are partiallydeglycosylated (or N-partially glycosylated polypeptides). For example,partially deglycosylated soluble PH20 polypeptides that retain all or aportion of the hyaluronidase activity of a fully glycosylatedhyaluronidase can be used in the compositions, combinations and/ormethods provided herein. Exemplary partially deglycosylatedhyalurodinases include soluble forms of a partially deglycosylated PH20polypeptides from any species, such as any set forth in any of SEQ IDNOS: 1, 2, 11, 25, 27, 29-32, 63, 65, and 101-102, or allelic variants,truncated variants, or other variants thereof. Such variants are knownto one of skill in the art, and include polypeptides having 60%, 70%,80%, 90%, 91%, 92%, 93%, 94%, 95% or more sequence identity to any ofSEQ ID NOS: 1, 2, 11, 25, 27, 29-32, 63, 65, and 101-102, or truncatedforms thereof. The partially deglycosylated hyaluronidases providedherein also include hybrid, fusion and chimeric partially deglycosylatedhyaluronidases, and partially deglycosylated hyaluronidase conjugates.

Glycosidases, or glycoside hydrolases, are enzymes that catalyze thehydrolysis of the glycosidic linkage to generate two smaller sugars. Themajor types of N-glycans in vertebrates include high mannose glycans,hybrid glycans and complex glycans. There are several glycosidases thatresult in only partial protein deglycosylation, including: EndoF1, whichcleaves high mannose and hybrid type glycans; EndoF2, which cleavesbiantennary complex type glycans; EndoF3, which cleaves biantennary andmore branched complex glycans; and EndoH, which cleaves high mannose andhybrid type glycans. Treatment of a hyaluronan degrading enzyme, such asa soluble hyaluronidase, such as a soluble PH20, with one or all ofthese glycosidases can result in only partial deglycosylation and,therefore, retention of hyaluronidase activity.

Partially deglycosylated hyaluronan degrading enzymes, such as partiallydeglycosylated soluble hyaluronidases, can be produced by digestion withone or more glycosidases, generally a glycosidase that does not removeall N-glycans but only partially deglycosylates the protein. Forexample, treatment of PH20 (e.g. a recombinant PH20 designated rHuPH20)with one or all of the above glycosidases (e.g. EndoF1, EndoF2 and/orEndoF3) results in partial deglycosylation. These partiallydeglycosylated PH20 polypeptides can exhibit hyaluronidase enzymaticactivity that is comparable to the fully glycosylated polypeptides. Incontrast, treatment of PH20 with PNGaseF, a glycosidase that cleaves allN-glycans, results in complete removal of all N-glycans and therebyrenders PH20 enzymatically inactive. Thus, although all N-linkedglycosylation sites (such as, for example, those at amino acids N82,N166, N235, N254, N368, and N393 of human PH20, exemplified in SEQ IDNO: 1) can be glycosylated, treatment with one or more glycosidases canrender the extent of glycosylation reduced compared to a hyaluronidasethat is not digested with one or more glycosidases.

The partially deglycosylated hyaluronan degrading enzymes, includingpartially deglycosylated soluble PH20 polypeptides, can have 10%, 20%,30%, 40%, 50%, 60%, 70% or 80% of the level of glycosylation of a fullyglycosylated polypeptide. In one example, 1, 2, 3, 4, 5 or 6 of theN-glycosylation sites corresponding to amino acids N82, N166, N235,N254, N368, and N393 of SEQ ID NO:1 are partially deglycosylated, suchthat they no longer contain high mannose or complex type glycans, butrather contain at least an N-acetylglucosamine moiety. In some examples,1, 2 or 3 of the N-glycosylation sites corresponding to amino acids N82,N166 and N254 of SEQ ID NO:1 are deglycosylated, that is, they do notcontain a sugar moiety. In other examples, 3, 4, 5, or 6 of theN-glycosylation sites corresponding to amino acids N82, N166, N235,N254, N368, and N393 of SEQ ID NO:1 are glycosylated. Glycosylated aminoacid residues minimally contain an N-acetylglucosamine moiety.Typically, the partially deglyclosylated hyaluronan degrading enzymes,including partially deglycosylated soluble PH20 polypeptides, exhibithyaluronidase activity that is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500%, 1000%or more of the hyaluronidase activity exhibited by the fullyglycosylated polypeptide.

iv. Modified (Polymer-Conjugated) Hyaluronan Degrading Enzymes

In one example, the provided compositions and combinations containhyaluronan degrading enzymes, in particular soluble hyaluronidases, thathave been modified by conjugation to one or more polymeric molecule(polymer), typically to increase the half-life of the hyaluronandegrading enzyme, for example, to promote prolonged/sustained treatmenteffects in a subject.

Covalent or other stable attachment (conjugation) of polymericmolecules, such as polyethylene glycol (PEGylation moiety (PEG)), to thehyaluronan degrading enzymes, such as hyaluronidases, impart beneficialproperties to the resulting hyaluronan degrading enzyme-polymercomposition. Such properties include improved biocompatibility,extension of protein (and enzymatic activity) half-life in the blood,cells and/or in other tissues within a subject, effective shielding ofthe protein from proteases and hydrolysis, improved biodistribution,enhanced pharmacokinetics and/or pharmacodynamics, and increased watersolubility.

Hence, in particular examples herein, the hyaluronan degrading enzyme isconjugated to a polymer. Exemplary of polymers are such as polyols (i.e.poly-OH), polyamines (i.e. poly-NH₂) and polycarboxyl acids (i.e.poly-COOH), and further heteropolymers i.e. polymers comprising one ormore different coupling groups e.g. a hydroxyl group and amine groups.Examples of suitable polymeric molecules include polymeric moleculesselected from among polyalkylene oxides (PAO), such as polyalkyleneglycols (PAG), including polyethylene glycols (PEG), methoxypolyethyleneglycols (mPEG) and polypropylene glycols, PEG-glycidyl ethers(Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG) branched polyethyleneglycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates,polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic acidanhydride, polystyrene-co-maleic acid anhydride, dextrans includingcarboxymethyl-dextrans, heparin, homologous albumin, celluloses,including methylcellulose, carboxymethylcellulose, ethylcellulose,hydroxyethylcellulose carboxyethylcellulose and hydroxypropylcellulose,hydrolysates of chitosan, starches such as hydroxyethyl-starches andhydroxypropyl-starches, glycogen, agaroses and derivatives thereof, guargum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acidhydrolysates and bio-polymers.

In particular, the polymer is a polyethylene glycol (PEG). Suitablepolymeric molecules for attachment to the hyaluronan degrading enzymes,including hyaluronidases, include, but are not limited to, polyethyleneglycol (PEG) and PEG derivatives such as methoxy-polyethylene glycols(mPEG), PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole(CDI-PEG), branched PEGs, and polyethylene oxide (PEO) (see e.g. Robertset al., Advanced Drug Delivery Review (2002) 54: 459-476; Harris andZalipsky, S (eds.) “Poly(ethylene glycol), Chemistry and BiologicalApplications” ACS Symposium Series 680, 1997; Mehvar et al., J. Pharm.Pharmaceut. Sci., 3(1):125-136, 2000; Harris, (2003) Nature Reviews DrugDiscovery 2:214-221; and Tsubery, (2004) J Biol. Chem 279(37):38118-24).The polymeric molecule can be of a molecular weight typically rangingfrom about 3 kDa to about 60 kDa. In some embodiments the polymericmolecule that is conjugated to a protein, such as rHuPH20, has amolecular weight of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 ormore than 60 kDa.

Various methods of modifying polypeptides by covalently attaching(conjugating) a PEG or PEG derivative (i.e. “PEGylation”) are known inthe art (see e.g., U.S. 2006/0104968; U.S. Pat. No. 5,672,662; U.S. Pat.No. 6,737,505; and U.S. 2004/0235734). Such techniques are describedelsewhere herein.

2. Pharmaceutical Compositions and Formulations

Provided herein are pharmaceutical compositions of anti-hyaluronanagents, for example, a hyaluronan-degrading enzyme or modified formthereof (e.g. a PEGylated hyaluronan-degrading enzymes, such asPEGylated hyaluronidases), for use in the treatment methods provided.Also provided herein are pharmaceutical compositions containing a secondagent that is used to treat a disease or disorder associated with ahyaluronan-associated disease or condition, such as cancer. Exemplary ofsuch agents include, but are not limited to, anti-cancer agentsincluding drugs, polypeptides, nucleic acids, antibodies, peptides,small molecules, gene therapy vector, viruses and other therapeutics.Anti-hyaluronan agents, for example, a hyaluronan-degrading enzyme ormodified form thereof (e.g. a PEGylated hyaluronan-degrading enzymes,such as PEGylated hyaluronidases or PEGPH20), can be co-formulated orco-administered with pharmaceutical formulations of such second agentsto enhance their delivery to desired sites or tissues within the bodyassociated with excess or accumulated hyaluronan.

Pharmaceutically acceptable compositions are prepared in view ofapprovals for a regulatory agency or other agency prepared in accordancewith generally recognized pharmacopeia for use in animals and in humans.The compounds can be formulated into any suitable pharmaceuticalpreparations for any of oral and intravenous administration such assolutions, suspensions, powders, or sustained release formulations.Typically, the compounds are formulated into pharmaceutical compositionsusing techniques and procedures well known in the art (see e.g., AnselIntroduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126).The formulation should suit the mode of administration.

In one example, pharmaceutical preparation can be in liquid form, forexample, solutions, syrups or suspensions. If provided in liquid form,the pharmaceutical preparations can be provided as a concentratedpreparation to be diluted to a therapeutically effective concentrationbefore use. Such liquid preparations can be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). In another example, pharmaceutical preparations can bepresented in lyophilized form for reconstitution with water or othersuitable vehicle before use.

Pharmaceutical compositions can include carriers such as a diluent,adjuvant, excipient, or vehicle with which the compositions (e.g.corticosteroid or anti-hyaluronan agent, such as a PEGylatedhyaluronan-degrading enzymes) are administered. Examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin. Such compositions will contain atherapeutically effective amount of the compound or agent, generally inpurified form or partially purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, andsesame oil. Water is a typical carrier. Saline solutions and aqueousdextrose and glycerol solutions also can be employed as liquid carriers,particularly for injectable solutions. Compositions can contain alongwith an active ingredient: a diluent such as lactose, sucrose, dicalciumphosphate, or carboxymethylcellulose; a lubricant, such as magnesiumstearate, calcium stearate and talc; and a binder such as starch,natural gums, such as gum acacia, gelatin, glucose, molasses,polyvinylpyrrolidine, celluloses and derivatives thereof, povidone,crospovidones and other such binders known to those of skill in the art.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, and ethanol. For example, suitable excipientsare, for example, water, saline, dextrose, glycerol or ethanol. Acomposition, if desired, also can contain other minor amounts ofnon-toxic auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, stabilizers, solubility enhancers, and other suchagents, such as for example, sodium acetate, sorbitan monolaurate,triethanolamine oleate and cyclodextrins.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers, which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN 80). A sequestering or chelating agent of metalions include EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles andsodium hydroxide, hydrochloric acid, citric acid or lactic acid for pHadjustment.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Preparationsfor intraprostatic administration include sterile solutions ready forinjection, sterile dry soluble products, such as lyophilized powders,ready to be combined with a solvent just prior to use, includinghypodermic tablets, sterile suspensions ready for injection, sterile dryinsoluble products ready to be combined with a vehicle just prior touse, sterile emulsions. The solutions can be either aqueous ornonaqueous.

3. Dosages and Administration

Typically, the dose of an anti-hyaluronan agent, for example, ahyaluronan-degrading enzyme, is one that also achieves a therapeuticeffect in the treatment of a hyaluronan associated disease or condition,such as cancer. Hence, compositions of an anti-hyaluronan agent, forexample a hyaluronan-degrading enzyme, are included in an amountsufficient to exert a therapeutically useful effect. The compositioncontaining the active agent can include a pharmaceutically acceptablecarrier. The compositions of an anti-hyaluronan agent also can include asecond therapeutic agent.

Therapeutically effective concentration of an anti-hyaluronan agent, forexample a hyaluronan-degrading enzyme, can be determined empirically bytesting the compounds in known in vitro and in vivo systems, such as theassays provided herein. For example, the concentration of ananti-hyaluronan agent, such as a hyaluronan-degrading enzyme or modifiedform thereof (e.g a PEGylated hyaluronan-degrading enzyme, such asPEGylated hyaluronidase) depends on absorption, inactivation andexcretion rates, the physicochemical characteristics, the dosageschedule, and amount administered as well as other factors known tothose of skill in the art. For example, it is understood that theprecise dosage and duration of treatment is a function of the tissuebeing treated, the disease or condition being treated, the route ofadministration, the patient or subject and the particularanti-hyaluronan agent and can be determined empirically using knowntesting protocols or by extrapolation from in vivo or in vitro test dataand/or can be determined from known dosing regimes of the particularagent. The amount of an anti-hyaluronan agent, for example ahyaluronan-degrading enzyme or modified form thereof (e.g. a PEGylatedhyaluronan-degrading enzyme, such as a PEGylated hyaluronidase), to beadministered for the treatment of a disease or condition, for example ahyaluronan-associated disease or condition such as an HA-rich tumor, canbe determined by standard clinical techniques. In addition, in vitroassays and animal models can be employed to help identify optimal dosageranges. The precise dosage, which can be determined empirically, candepend on the particular enzyme, the route of administration, the typeof disease to be treated and the seriousness of the disease.

For example, methods of using anti-hyaluronan agents, such ashyaluronan-degrading enzymes or modified forms thereof (e.g. PEGylatedforms) for treatment of hyaluronan-associated diseases and conditionsare well known in the art (see e.g. U.S. published application No.20100003238 and International published PCT Appl. No. WO 2009/128917).Thus, dosages of an anti-hyaluronan agent, such as ahyaluronan-degrading enzyme for example a hyaluronidase, can be chosenbased on standard dosing regimes for that agent under a given route ofadministration.

Examples of effective amounts of an anti-hyaluronan agent for treatmentof a hyaluronan-associated disease or condition is a dose ranging from0.01 μg to 100 g per kg of body weight. For example, an effective amountof an anti-hyaluronan agent is a dose ranging from 0.01 μg to 100 mg perkg of body weight, such as 0.01 μg to 1 mg per kg of body weight, 1 μgto 100 μg per kg of body weight, 1 μg to 10 μg per kg of body weight or0.01 mg to 100 mg per kg of body weight. For example, effective amountsinclude at least or about at least or about or 0.01 μg, 0.05, 0.1, 0.5,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or1000 μg/kg body weight. Other examples of effective amounts include0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 g/kg bodyweight. For example, an anti-hyaluronan agent, such as ahyaluronan-degrading enzyme for example a hyaluronidase (e.g. aPEGylated hyaluronidase such as a PEGPH20), can be administered at orabout 0.1 μg/kg to 1 mg/kg, for example 0.5 μg/kg to 100 μg/kg, 0.75μg/kg to 15 μg/kg, 0.75 μg/kg to 7.5 μg/kg or 1.0 μg/kg to 3.0 μg/kg. Inother examples, an anti-hyaluronan agent such as a hyaluronan-degradingenzyme for example a hyaluornidase (e.g. a PEGylated hyaluronidase suchas a PEGPH20), can be administered at or 1 mg/kg to 500 mg/kg, forexample, 100 mg/kg to 400 mg/kg, such as 200 mg/kg. For example,compositions contain 0.5 mg to 100 grams of anti-hyaluronan agent, forexample, 20 μg to 1 mg, such as 100 μg to 0.5 mg or can contain 1 mg to1 gram, such as 5 mg to 500 mg.

For example, agents and treatments for treatment ofhyaluronan-associated diseases and conditions, such as anti-canceragents, are well known in the art (see e.g. U.S. published applicationNo. 20100003238 and International published PCT Appl. No. WO2009/128917). Thus, dosages of a hyaluronan-degrading enzyme, forexample a hyaluronidase, or other second agents in a composition can bechosen based on standard dosing regimes for that agent under a givenroute of administration.

Examples of effective amounts of a hyaluronan-degrading enzyme is a doseranging from 0.01 μg to 100 g per kg of body weight. For example, aneffective amount of a hyaluronan-degrading enzyme is a dose ranging from0.01 μg to 100 mg per kg of body weight, such as 0.01 μg to 1 mg per kgof body weight, 1 μg to 100 μg per kg of body weight, 1 μg to 10 μg perkg of body weight or 0.01 mg to 100 mg per kg of body weight. Forexample, effective amounts include at least or about at least or aboutor 0.01 μg, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400,500, 600, 700, 800, 900 or 1000 μg/kg body weight. Other examples ofeffective amounts include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 100 g/kg body weight. For example, a hyaluronan-degradingenzyme for example a hyaluronidase (e.g. a PEGylated hyaluronidase suchas a PEGPH20), can be administered at or about 0.1 μg/kg to 1 mg/kg, forexample 0.5 μg/kg to 100 μg/kg, 0.75 μg/kg to 15 μg/kg, 0.75 μg/kg to7.5 μg/kg or 1.0 μg/kg to 3.0 μg/kg. In other examples, ahyaluronan-degrading enzyme for example a hyaluornidase (e.g. aPEGylated hyaluronidase such as a PEGPH20), can be administered at or 1mg/kg to 500 mg/kg, for example, 100 mg/kg to 400 mg/kg, such as 200mg/kg. Generally, compositions contain 0.5 mg to 100 grams of ahyaluronan-degrading enzyme, for example, 20 μg to 1 mg, such as 100 μgto 0.5 mg or can contain 1 mg to 1 gram, such as 5 mg to 500 mg.

The dose or compositions can be for single dosage administration or formultiple dosage administration. The dose or composition can beadministered in a single administration once, several times a week,twice weekly, every 15 days, 16 days, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 days, once monthly, several times a year oryearly. In other examples, the dose or composition an be divided up andadministered once, several times a week, twice weekly, every 15 days, 16days, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days,once monthly, several times a year or yearly. Hyaluronan-degradingenzyme compositions can be formulated as liquid compositions or can belyophilized. The compositions also can be formulated as a tablet orcapsule.

Provided below is description of dosages and dosage regimines ofexemplary hyaluronan-degrading enzymes conjugated to a polymer (e.g.PEGylated) for use in the methods herein. The hyaluronan-degradingenzymes can be used alone in a single agent therapy or in combinationwith other agents for use in treating an HA-associated disease orcondition, such as cancer. As discussed elsewhere herein, in particularexamples of the methods and uses herein, the agents can be administeredin combination with a corticosteroid in order to ameliorate aside-effect associated with treatment of the anti-hyaluronan-agent.

a. Administration of a PEGylated Hyaluronan-Degrading Enzyme

A hyaluronan-degrading enzyme, such as a PEGylated hyaluronan-degradingenzyme (e.g. a hyaluronidase), can be administered systemically, forexample, intravenously (IV), intramuscularly, or by any another systemicroute. In particular examples, lower doses can be given locally. Forexample, local administration of a hyaluronan-degrading enzyme, such asa PEGylated hyaluronan-degrading enzyme for example a PEGylatedhyaluronidase (e.g. PH20) includes intratumoral administration, arterialinjection (e.g. hepatic artery), intraperitoneal administration,intravesical administration and other local routes used for cancertherapy that can increase local action at a lower absolute dose.

Exemplary dosage range is at or about 0.3 Units/kg to 320,000 Units/kg,such as 10 Units/kg to 320,000 Units/kg of a PEGylated hyaluronidase, ora functionally equivalent amount of another PEGylatedhyaluronan-degrading enzyme. It is understood herein that a unit ofactivity is normalized to a standard activity, for example, an activityas measured in a microturbidity assay assaying hyaluronidase activity. APEGylated soluble hyaluronidase can exhibit lower activity per mg oftotal protein, i.e. exhibits a lower specific activity, compared to anative soluble hyaluronidase not so conjugated. For example, anexemplary rHuPH20 preparation exhibits a specific activity of 120,000Units/mg, while a PEGylated form of rHuPH20 exhibits a specific activityof at or about 32,000 Units/mg. Typically, a PEGylated form of ahyaluronan-degrading enzyme, such as a hyaluronidase for examplerHuPH20, exhibits a specific activity within the range of between at orabout 18,000 and at or about 45,000 U/mg. In one example, thePEG-hyaluronan-degrading enzyme can be provided as a stock solution forexample, at 3.5 mg/mL at 112,000 U/mL (˜32,000 U/mg), with a PEG toprotein molar ratio between 5:1 and 9:1, for example, 7:1, or can beprovided in a less concentrated form. For purposes herein, dosages canbe with reference to Units.

For example, PEGylated hyaluronan-degrading enzyme, such as ahyaluronidase, for example PEGPH20, can be administered intravenouslytwice weekly, once weekly or once every 21 days. Typically, thePEGylated hyaluronan-degrading enzyme is administered twice weekly. Thecycle of administration can be for a defined period, generally for 3weeks or 4 weeks. The cycle of administration can be repeated in adosage regime for more than one month, 2 months, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1year or more. Generally, the cycle of administration is repeated at thediscretion of a treating physician, and can depend on factors such asremission of the disease or condition, severity of the disease orcondition, adverse events and other factors. In other examples, insubsequent cycles of administration, the hyaluronan-degrading enzyme canbe administered less frequently. For example, in a first cycle thehyaluronan-degrading enzyme is administered twice weekly for four weeks,and in subsequent cycles of administration the hyaluronan-degradingenzyme is administered once weekly or once every two weeks, once every 3weeks (e.g. once every 21 days) or once every 4 weeks. As describedherein, the dose or dosing regime of corticosteroid is dependent on thedosing regime of hyaluronan-degrading enzyme.

While dosages can vary depending on the disease and patient, thehyaluronan-degrading enzyme, such as a PEGylated hyaluronidase, isgenerally administered in an amount that is or is about in the range offrom 0.01 μg/kg, such as 0.0005 mg/kg (0.5 μg/kg) to 10 mg/kg (320,000U/kg), for example, 0.02 mg/kg to 1.5 mg/kg, for example, 0.05 mg/kg.The PEGylated hyaluronidase can be administered, for example, at adosage of at or about 0.0005 mg/kg (of the subject), 0.0006 mg/kg,0.0007 mg/kg, 0.0008 mg/kg, 0.0009 mg/kg, 0.001 mg/kg, 0.0016 mg/kg,0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.016 mg/kg, 0.02 mg/kg,0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg,0.09 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.30 mg/kg,0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg·kg, 0.6 mg/kg,0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg,2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9mg/kg, 9.5 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, or more is administered, to anaverage adult human subject, typically weighing about 70 kg to 75 kg. Inparticular examples, the hyaluronan-degrading enzyme is administered inlower amounts such as less than 20 μg/kg, for example 0.01 μg/kg to 15μg/kg, 0.05 μg/kg to 10 μg/kg, 0.75 μg/kg to 7.5 μg/kg or 1.0 μg/kg to3.0 μg/kg.

A hyaluronan-degrading enzyme, such as a PEGylated hyaluronidase (e.g.PH20), provided herein, for example, PEGPH20, can be administered at orabout 1 Unit/kg to 800,000 Units/kg, such as 10 to 800,000 Units/kg, 10to 750,000 Units/kg, 10 to 700,000 Units/kg, 10 to 650,000 Units/kg, 10to 600,000 Units/kg, 10 to 550,000 Units/kg, 10 to 500,000 Units/kg, 10to 450,000 Units/kg, 10 to 400,000 Units/kg, 10 to 350,000 Units/kg, 10to 320,000 Units/kg, 10 to 300,000 Units/kg, 10 to 280,000 Units/kg, 10to 260,000 Units/kg, 10 to 240,000 Units/kg, 10 to 220,000 Units/kg, 10to 200,000 Units/kg, 10 to 180,000 Units/kg, 10 to 160,000 Units/kg, 10to 140,000 Units/kg, 10 to 120,000 Units/kg, 10 to 100,000 Units/kg, 10to 80,000 Units/kg, 10 to 70,000 Units/kg, 10 to 60,000 Units/kg, 10 to50,000 Units/kg, 10 to 40,000 Units/kg, 10 to 30,000 Units/kg, 10 to20,000 Units/kg, 10 to 15,000 Units/kg, 10 to 12,800 Units/kg, 10 to10,000 Units/kg, 10 to 9,000 Units/kg, 10 to 8,000 Units/kg, 10 to 7,000Units/kg, 10 to 6,000 Units/kg, 10 to 5,000 Units/kg, 10 to 4,000Units/kg, 10 to 3,000 Units/kg, 10 to 2,000 Units/kg, 10 to 1,000Units/kg, 10 to 900 Units/kg, 10 to 800 Units/kg, 10 to 700 Units/kg, 10to 500 Units/kg, 10 to 400 Units/kg, 10 to 300 Units/kg, 10 to 200Units/kg, 10 to 100 Units/kg, 16 to 600,000 Units/kg, 16 to 500,000Units/kg, 16 to 400,000 Units/kg, 16 to 350,000 Units/kg, 16 to 320,000Units/kg, 16 to 160,000 Units/kg, 16 to 80,000 Units/kg, 16 to 40,000Units/kg, 16 to 20,000 Units/kg, 16 to 16,000 Units/kg, 16 to 12,800Units/kg, 16 to 10,000 Units/kg, 16 to 5,000 Units/kg, 16 to 4,000Units/kg, 16 to 3,000 Units/kg, 16 to 2,000 Units/kg, 16 to 1,000Units/kg, 16 to 900 Units/kg, 16 to 800 Units/kg, 16 to 700 Units/kg, 16to 500 Units/kg, 16 to 400 Units/kg, 16 to 300 Units/kg, 16 to 200Units/kg, 16 to 100 Units/kg, 160 to 12,800 Units/kg, 160 to 8,000Units/kg, 160 to 6,000 Units/kg, 160 to 4,000 Units/kg, 160 to 2,000Units/kg, 160 to 1,000 Units/kg, 160 to 500 Units/kg, 500 to 5000Units/kg, 1000 to 100,000 Units/kg or 1000 to 10,000 Units/kg, of themass of the subject to whom it is administered. In some examples, ahyaluronan-degrading enzyme, such as a PEGylated hyaluronidase (e.g.PH20), provided herein, for example, PEGPH20, can be administered at orabout 1 Unit/kg to 1000 Units/kg, 1 Units/kg to 500 Units/kg or 10Units/kg to 50 Units/kg.

Generally, where the specific activity of the PEGylated hyaluronidase isor is about 18,000 U/mg to 45,000 U/mg, generally at or about 1 Units/kg(U/kg), 2 U/kg, 3 U/kg, 4 U/kg, 5 U/kg, 6 U/kg, 7 U/kg, 8 U/kg, 8 U/kg10 U/kg, 16 U/kg, 32 U/kg, 64 U/kg, 100 U/kg, 200 U/kg, 300 U/kg, 400U/kg, 500 U/kg, 600 U/kg, 700 U/kg, 800 U/kg, 900 U/kg, 1,000 U/kg,2,000 U/kg, 3,000 U/kg, 4,000 U/kg, 5,000 U/kg, 6,000 U/kg, 7,000 U/kg,8,000 U/kg, 9,000 U/kg, 10,000 U/kg, 12,800 U/kg, 20,000 U/kg, 32,000U/kg, 40,000 U/kg, 50,000 U/kg, 60,000 U/kg, 70,000 U/kg, 80,000 U/kg,90,000 U/kg, 100,000 U/kg, 120,000 U/kg, 140,000 U/kg, 160,000 U/kg,180,000 U/kg, 200,000 U/kg, 220,000 U/kg, 240,000 U/kg, 260,000 U/kg,280,000 U/kg, 300,000 U/kg, 320,000 U/kg, 350,000 U/kg, 400,000 U/kg,450,000 U/kg, 500,000 U/kg, 550,000 U/kg, 600,000 U/kg, 650,000 U/kg,700,000 U/kg, 750,000 U/kg, 800,000 U/kg or more, per mass of thesubject, is administered.

In some aspects, the PEGylated hyaluronan-degrading enzyme is formulatedand dosed to maintain at least 3 U/mL of the PEGylated hyaluronidase inthe plasma (see e.g. published U.S. Patent App. No. US20100003238 andpublished International Patent App. No. WO2009128917). For example, thePEGylated soluble hyaluronidase is formulated for systemicadministration in a sufficient amount to maintain at least or about 3U/mL in the plasma, generally 3 U/mL-12 U/mL or more, for example, fromat least or about or at a level of 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8U/mL, 9 U/mL, 10 U/mL, 11 U/mL, 12 U/mL, 13 U/mL, 14 U/mL, 15 U/mL, 16U/mL, 17 U/mL, 18 U/mL, 19 U/mL, 20 U/mL, 25 U/mL, 30 U/mL, 35 U/mL, 40U/mL, 45 U/mL, 50 U/mL or more. Generally, for purposes herein tomaintain at least 3 U/mL of the hyaluronidase in plasma, at or about0.02 mg/kg (of the subject), 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.5mg/kg, 0.55 mg·kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg or more isadministered. Generally, where the specific activity of the modifiedhyaluronidase is or is about 20,000 U/mg to 60,000 U/mg, generally at orabout 35,000 U/mg, 60,000 U; 70,000 U; 80,000 U; 90,000 U; 100,000 U;200,000 U; 300,000 U; 400,000 U; 500,000 U; 600,000 U; 700,000 U;800,000 U; 900,000 U; 1,000,000 U; 1,500,000 U; 2,000,000 U; 2,500,000U; 3,000,000 U; 3,500,000 U; 4,000,000 U or more is administered. Tomaintain such levels, administration can be daily, several times a week,twice weekly, weekly or monthly.

It is within the level of one of skill in the art to determine theamounts of PEGylated hyaluron degrading enzyme, for example, PEGylatedPH20, to maintain at least 3 U/mL of the hyaluronidase in the blood. Thelevel of hyaluronidase in the blood can be monitored over time in orderto ensure that a sufficient amount of the hyaluronidase is present inthe blood. Any assay known to one of skill in the art to measure thehyaluronidase in the plasma can be performed. For example, amicroturbidity assay or enzymatic assay described in the Examples hereincan be performed on protein in plasma. It is understood that plasmanormally contains hyaluronidase enzymes. Such plasma hyaluronidaseenzymes typically have activity at an acidic pH (U.S. Pat. No.7,105,330). Hence, before treatment of with a modified enzyme, theplasma levels of hyaluronidase should be determined and used as abaseline. Subsequent measurements of plasma hyaluronidase levels aftertreatment can be compared to the levels before treatments.Alternatively, the assay can be performed under pH conditions thatsuppress endogenous lysosomal hyaluronidase activity in plasma, whichnormally exhibits activity at acidic pH. Thus, where the modifiedsoluble hyaluronidase is active at neutral pH (e.g. human PH20), onlythe level of the modified neutral-active soluble hyaluronidase ismeasured.

In other examples, the PEGylated hyaluronan-degrading enzyme isformulated and administered at a lower dose, which is found herein tohave therapeutic effects to treat a hyaluronan-associated disease orconditions absent a detectable level of hyaluronidase maintained in theblood. For example, the PEGylated soluble hyaluronidase is administeredin an amount that is less than 20 μg/kg, for example 0.01 μg/kg to 15μg/kg, 0.05 μg/kg to 10 μg/kg, 0.75 μg/kg to 7.5 μg/kg or 1.0 μg/kg to3.0 μg/kg, such as at or about 0.01 μg/kg (of the subject), 0.02 μg/kg,0.03 μg/kg, 0.04 μg/kg, 0.05 μg/kg, 1.0 μg/kg, 1.5 μg/kg, 2.0 μg/kg, 2.5μg/kg, 3.0 μg/kg, 3.5 μg/kg, 4.0 μg/kg, 4.5 μg/kg, 5.0 μg/kg, 5.5 μg/kg,6.0 μg/kg, 7.0 μg/kg, 7.5 μg/kg, 8.0 μg/kg, 9.0 μg/kg, 10.0 μg/kg, 12.5μg/kg or 15 μg/kg. Generally, where the specific activity of themodified hyaluronidase is or is about 20,000 U/mg to 60,000 U/mg,generally at or about 35,000 U/mg, 200 Units to 50,000 (U) isadministered, such as 200 U, 300 U; 400 U; 500 U; 600 U; 700 U; 800 U;900 U; 1,000 U; 1250 U; 1500 U; 2000 U; 3000 U; 4000 U; 5,000 U; 6,000U; 7,000 U; 8,000 U; 9,000 U; 10,000 U; 20,000 U; 30,000 U; 40,000 U; or50,000 U is administered. To maintain such levels, administration can bedaily, several times a week, twice weekly, weekly or monthly.

Typically, volumes of injections or infusions of PEGylated hyaluronidasecontemplated herein are from at or about 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL,5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40 mL, 50 mLor more. The PEGylated hyaluronan-degrading enzyme, such as a PEGylatedhyaluronidase can be provided as a stock solution at or about 50 U/mL,100 U/mL, 150 U/mL, 200 U/mL, 400 U/mL or 500 U/mL (or a functionallyequivalent amount) or can be provided in a more concentrated form, forexample at or about 1000 U/mL, 2000 Units/mL, 3000 U/mL, 4000 U/mL, 5000U/mL, 6000 U/mL, 7000 U/mL, 8000 U/mL, 9000 U/mL, 10,000 U/mL, 11,000U/mL, 12,000 U/mL, or 12,800 U/mL, for use directly or for dilution tothe effective concentration prior to use. The volume of PEGylatedhyaluronan-degrading enzyme, such as PEGylated hyaluronidase,administered is a function of the dosage required, but can be varieddepending on the concentration of a hyaluronan-degrading enzyme, such assoluble hyaluronidase, stock formulation available. For example, it iscontemplated herein that the PEGylated hyaluronan-degrading enzyme, suchas PEGylated hyaluronidase, is not administered in volumes greater thanabout 50 mL, and typically is administered in a volume of 5-30 mL,generally in a volume that is not greater than about 10 mL. ThePEGylated hyaluronan-degrading enzyme, such as a PEGylatedhyaluronidase, can be provided as a liquid or lyophilized formulation.Lyophilized formulations are ideal for storage of large unit doses ofPEGylated hyaluronan-degrading enzymes.

4. Combination Treatments

Anti-hyaluronan agents, such as a hyaluronan-degrading enzymes ormodified form thereof (e.g. a PEGylated hyaluronan-degrading enzyme orPEGylated hyaluronaidase such as PEGPH20) can be administered in acombination treatment, for example, for the treatment of ahyaluronan-associated disease or condition, such as cancer. Compositionsof an anti-hyaluronan agent can be co-formulated or co-administeredtogether with, prior to, intermittently with, or subsequent to, othertherapeutic or pharmacologic agents or treatments, such as procedures,for example, agents or treatments to treat a hyaluronan associateddisease or condition, for example hyaluronan-associated cancers. Suchagents include, but are not limited to, other biologics, anti-canceragents, small molecule compounds, dispersing agents, anesthetics,vasoconstrictors and surgery, and combinations thereof. Such otheragents and treatments that are available for the treatment of a diseaseor condition, including all those exemplified herein, are known to oneof skill in the art or can be empirically determined.

A preparation of a second agent or agents or treatment or treatments canbe administered at once, or can be divided into a number of smallerdoses to be administered at intervals of time. Selected agent/treatmentpreparations can be administered in one or more doses over the course ofa treatment time for example over several hours, days, weeks, or months.In some cases, continuous administration is useful. It is understoodthat the precise dosage and course of administration depends on theindication and patient's tolerability. Generally, dosing regimes forsecond agents/treatments herein are known to one of skill in the art.

In one example, an anti-hyaluronan agent, for example ahyaluronan-degrading enzyme or modified form thereof conjugated to apolymer (e.g. a PEGylated hyaluronan-degrading enzyme, such as PEGylatedhyaluronidase), is administered with a second agent or treatment fortreating the disease or condition. In one example, an anti-hyaluronanagent, for example a hyaluronan-degrading enzyme or a modified formthereof conjugated to a polymer (e.g. a PEGylated hyaluronan-degradingenzyme) and second agent or treatment can be co-formulated andadministered together. In another example, an anti-hyaluronan agent, forexample a hyaluronan-degrading enzyme or modified form thereofconjugated to a polymer (e.g. a PEGylated hyaluronan-degrading enzyme,such as PEGylated hyaluronidase) is administered subsequently,intermittently or simultaneously with the second agent or treatmentpreparation. Generally, an anti-hyaluronan agent, for example ahyaluronan-degrading enzyme (e.g. a PEGylated hyaluronan-degradingenzyme) is administered prior to administration of the second agent ortreatment preparation to permit the agent to reduce the level or amountof tissue- or cell-associated hyaluronan. For example, ahyaluronan-degrading enzyme, for example a PEGylatedhyaluronan-degrading enzyme, is administered prior to a second agent ortreatment to permit the enzyme to reduce or degrade the hyaluronic acidin a cell, tissue or fluid of the subject, such as, for example, theinterstitial space, extracellular matrix, tumor tissue, blood or othertissue. For example, an anti-hyaluronan agent, such as ahyaluronan-degrading enzyme or modified form thereof conjugated to apolymer (e.g. a PEGylated hyaluronan-degrading enzyme, such as solublehyaluronidase) can be administered 0.5 minutes, 1 minute, 2 minutes, 3minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour or more prior toadministration of the second agent preparation. In some examples, ananti-hyaluronan agent, for example a hyaluronan-degrading enzyme ormodified form thereof conjugated to a polymer (e.g. a PEGylatedhyaluronan-degrading enzyme) is administered together with the secondagent preparation. As will be appreciated by those of skill in the art,the desired proximity of co-administration depends in significant partin the effective half lives of the agents in the particular tissuesetting, and the particular disease being treated, and can be readilyoptimized by testing the effects of administering the agents at varyingtimes in suitable models, such as in suitable animal models. In somesituations, the optimal timing of administration of the anti-hyaluronanagent, for example a hyaluronan-degrading enzyme or modified formthereof conjugated to a polymer (e.g. a PEGylated hyaluronan-degradingenzyme, such as a PEGylated hyaluronidase) will exceed 60 minutes.

For example, an anti-hyaluronan agent, for example ahyaluronan-degrading enzyme, can be administered in conjunction withanti-cancer agents (see e.g. U.S. Publication No. US2010-0003238). Theanticancer agent(s) or treatment(s) for use in combination with ahyaluronan-degrading enzyme include, but are not limited to, surgery,radiation, drugs, chemotherapeutics, polypeptides, antibodies, peptides,small molecules or gene therapy vectors, viruses or DNA.

In other examples, the methods of treatment provided herein includemethods of administering one or more additional anti-hyaluronan agentsfor therapy in addition to a hyaluronan-degrading enzyme.Anti-hyaluronan agents include any agent that reduces or eliminates theaccumulation or HA in a tumor. Such agents include, but are not limitedto, the hyaluronan-degrading enzymes described herein and also agentsthat inhibit synthesis of HA. For example, anti-hyaluronan agents thatinhibit hyaluronan synthesis include antisense or sense moleculesagainst an has gene. Such antisense or sense inhibition is typicallybased upon hydrogen bonding-based hybridization of oligonucleotidestrands or segments such that at least one strand or segment is cleaved,degraded or otherwise rendered inoperable. In other examples,post-transcriptional gene silencing (PTGS), RNAi, ribozymes and DNAzymescan be employed. It is within the level of one skill in the art togenerate such constructs based on the sequence of HAS1 (set forth in SEQID NO: 195), HAS2 (set forth in SEQ ID NO:196) or HAS3 (set forth in SEQID NO:197 or 198). It is understood in the art that the sequence of anantisense or sense compound need not be 100% complementary to that ofits target nucleic acid to be specifically hybridizable. Anoligonucleotide may hybridize over one or more segments such thatintervening or adjacent segments are not involved in the hybridizationevent (e.g. a loop structure or hairpin structure). Generally, theantisense or sense compounds have at least 70% sequence complementarityto a target region within the target nucleic acid, for example, 75% to100% complementarity, such as 75%, 80%, 85%, 90%, 95% or 100%. Exemplarysense or antisense molecules are known in the art (see e.g. Chao et al.(2005) J. Biol. Chem. 280:27513-27522; Simpson et al. (2002) J. Biol.Chem. 277:10050-10057; Simpson et al. (2002) Am. J Path. 161:849;Nishida et al. (1999) J. Biol. Chem. 274:21893-21899; Edward et al.(2010) British J Dermatology 162:1224-1232; Udabage et al. (2005) CancerRes. 65:6139; and published U.S. Patent application No. US20070286856).Another exemplary anti-hyaluronan agent that is an HA synthesisinhibitor is 4-Methylumbelliferone (4-MU; 7-hydroxy-4-methylcoumarin) ora derivative thereof 4-MU acts by reducing the UDP-GlcUA precursor poolthat is required for HA synthesis. Further exemplary anti-hyaluronanagents are tyrosine kinase inhibitors, such as Leflunomide (Arava),genistein or erbstatin.

In some examples, a corticosteroid can be administered to ameliorateside effects or adverse events of a hyaluronan-degrading enzyme in thecombination therapy (see e.g. U.S. patent application Ser. No.13/135,817). In some examples, the glucocorticoid is selected from amongcortisones, dexamethasones, hydrocortisones, methylprednisolones,prednisolones and prednisones. In a particular example, theglucocorticoid is dexamethasone. Typically, the corticosteroid isadministered orally, although any method of administration of thecorticosteroid is contemplated. Typically, the glucocorticoid isadministered at an amount between at or about 0.4 and 20 mgs, forexample, at or about 0.4 mgs, 0.5 mgs, 0.6 mgs, 0.7 mgs, 0.75 mgs, 0.8mgs, 0.9 mgs, 1 mg, 2 mgs, 3 mgs, 4 mgs, 5 mgs, 6 mgs, 7 mgs, 8 mgs, 9mgs, 10 mgs, 11 mgs, 12 mgs, 13 mgs, 14 mgs, 15 mgs, 16 mgs, 17 mgs, 18mgs, 19 mgs or 20 mgs per dose.

F. METHODS OF PRODUCING NUCLEIC ACIDS AND ENCODED POLYPEPTIDES OFHYALURONAN BINDING PROTEINS AND HYALURONAN-DEGRADING ENZYMES

Polypeptides of a hyaluronan binding protein for use in the compositionsand methods provided or a hyaluronan-degrading enzyme, such as a solublehyaluronidase, for treatment set forth herein, can be obtained bymethods well known in the art for protein purification and recombinantprotein expression. Any method known to those of skill in the art foridentification of nucleic acids that encode desired genes can be used.Any method available in the art can be used to obtain a full length(i.e., encompassing the entire coding region) cDNA or genomic DNA cloneencoding a hyaluronan binding protein or a hyaluronidase, such as from acell or tissue source. Modified or variant hyaluronan binding proteinsor hyaluronidases, can be engineered from a wildtype polypeptide, suchas by site-directed mutagenesis.

Polypeptides can be cloned or isolated using any available methods knownin the art for cloning and isolating nucleic acid molecules. Suchmethods include PCR amplification of nucleic acids and screening oflibraries, including nucleic acid hybridization screening,antibody-based screening and activity-based screening.

Methods for amplification of nucleic acids can be used to isolatenucleic acid molecules encoding a desired polypeptide, including forexample, polymerase chain reaction (PCR) methods. A nucleic acidcontaining material can be used as a starting material from which adesired polypeptide-encoding nucleic acid molecule can be isolated. Forexample, DNA and mRNA preparations, cell extracts, tissue extracts,fluid samples (e.g. blood, serum, saliva), samples from healthy and/ordiseased subjects can be used in amplification methods. Nucleic acidlibraries also can be used as a source of starting material. Primers canbe designed to amplify a desired polypeptide. For example, primers canbe designed based on expressed sequences from which a desiredpolypeptide is generated. Primers can be designed based onback-translation of a polypeptide amino acid sequence. Nucleic acidmolecules generated by amplification can be sequenced and confirmed toencode a desired polypeptide.

Additional nucleotide sequences can be joined to a polypeptide-encodingnucleic acid molecule, including linker sequences containing restrictionendonuclease sites for the purpose of cloning the synthetic gene into avector, for example, a protein expression vector or a vector designedfor the amplification of the core protein coding DNA sequences.Furthermore, additional nucleotide sequences specifying functional DNAelements can be operatively linked to a polypeptide-encoding nucleicacid molecule. Examples of such sequences include, but are not limitedto, promoter sequences designed to facilitate intracellular proteinexpression, and secretion sequences, for example heterologous signalsequences, designed to facilitate protein secretion. Such sequences areknown to those of skill in the art. Additional nucleotide residuessequences such as sequences of bases specifying protein binding regionsalso can be linked to enzyme-encoding nucleic acid molecules. Suchregions include, but are not limited to, sequences of residues thatfacilitate or encode proteins that facilitate uptake of an enzyme intospecific target cells, or otherwise alter pharmacokinetics of a productof a synthetic gene. For example, enzymes can be linked to PEG moieties.

In addition, tags or other moieties can be added, for example, to aid indetection or affinity purification of the polypeptide. For example,additional nucleotide residues sequences such as sequences of basesspecifying an epitope tag or other detectable marker also can be linkedto enzyme-encoding nucleic acid molecules. Exemplary of such sequencesinclude nucleic acid sequences encoding a His tag (e.g., 6×His, HHHHHH;SEQ ID NO:54) or Flag Tag (DYKDDDDK; SEQ ID NO:55).

The identified and isolated nucleic acids can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art can be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene, La Jolla, Calif.). Other expression vectors include theHZ24 expression vector exemplified herein. The insertion into a cloningvector can, for example, be accomplished by ligating the DNA fragmentinto a cloning vector which has complementary cohesive termini.Insertion can be effected using TOPO cloning vectors (Invitrogen,Carlsbad, Calif.). If the complementary restriction sites used tofragment the DNA are not present in the cloning vector, the ends of theDNA molecules can be enzymatically modified. Alternatively, any sitedesired can be produced by ligating nucleotide sequences (linkers) ontothe DNA termini; these ligated linkers can contain specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. In an alternative method, the cleaved vector andprotein gene can be modified by homopolymeric tailing. Recombinantmolecules can be introduced into host cells via, for example,transformation, transfection, infection, electroporation andsonoporation, so that many copies of the gene sequence are generated.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated protein gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene can be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

1. Vectors and Cells

For recombinant expression of one or more of the desired proteins, suchas any hyaluronan binding protein or hyaluronan-degrading enzymedescribed herein, the nucleic acid containing all or a portion of thenucleotide sequence encoding the protein can be inserted into anappropriate expression vector, i.e., a vector that contains thenecessary elements for the transcription and translation of the insertedprotein coding sequence. The necessary transcriptional and translationalsignals also can be supplied by the native promoter for enzyme genes,and/or their flanking regions.

Also provided are vectors that contain a nucleic acid encoding theenzyme. Cells containing the vectors also are provided. The cellsinclude eukaryotic and prokaryotic cells, and the vectors are anysuitable for use therein.

Prokaryotic and eukaryotic cells, including endothelial cells,containing the vectors are provided. Such cells include bacterial cells,yeast cells, fungal cells, Archea, plant cells, insect cells and animalcells. The cells are used to produce a protein thereof by growing theabove-described cells under conditions whereby the encoded protein isexpressed by the cell, and recovering the expressed protein. Forpurposes herein, for example, the enzyme can be secreted into themedium.

Provided are vectors that contain a sequence of nucleotides that encodesthe hyaluronan-degrading enzyme polypeptide, in some examples a solublehyaluronidase polypeptide, coupled to the native or heterologous signalsequence, as well as multiple copies thereof. The vectors can beselected for expression of the enzyme protein in the cell or such thatthe enzyme protein is expressed as a secreted protein.

A variety of host-vector systems can be used to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g. vaccinia virus, adenovirus and otherviruses); insect cell systems infected with virus (e.g. baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements can be used.

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector can be used to construct expression vectorscontaining a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods can include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding protein, or domains,derivatives, fragments or homologs thereof, can be regulated by a secondnucleic acid sequence so that the genes or fragments thereof areexpressed in a host transformed with the recombinant DNA molecule(s).For example, expression of the proteins can be controlled by anypromoter/enhancer known in the art. In a specific embodiment, thepromoter is not native to the genes for a desired protein. Promoterswhich can be used include but are not limited to the SV40 early promoter(Bernoist and Chambon, Nature 290:304-310 (1981)), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al. Cell 22:787-797 (1980)), the herpes thymidine kinase promoter(Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), theregulatory sequences of the metallothionein gene (Brinster et al.,Nature 296:39-42 (1982)); prokaryotic expression vectors such as theβ-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA80:21-25 (1983)); see also Gilbert and Villa-Komaroff “Useful Proteinsfrom Recombinant Bacteria” Scientific American 242:74-94 (1980)); plantexpression vectors containing the nopaline synthetase promoter(Herrera-Estrella et al., Nature 303:209-213 (1984)) or the cauliflowermosaic virus 35S RNA promoter (Gardner et al., Nucleic Acids Res. 9:2871(1981)), and the promoter of the photosynthetic enzyme ribulosebisphosphate carboxylase (Herrera-Estrella et al., Nature 310:115-120(1984)); promoter elements from yeast and other fungi such as the Gal4promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinasepromoter, the alkaline phosphatase promoter, and the following animaltranscriptional control regions that exhibit tissue specificity and havebeen used in transgenic animals: elastase I gene control region which isactive in pancreatic acinar cells (Swift et al., Cell 38:639-646 (1984);Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986);MacDonald, Hepatology 7:425-515 (1987)); insulin gene control regionwhich is active in pancreatic beta cells (Hanahan et al., Nature315:115-122 (1985)), immunoglobulin gene control region which is activein lymphoid cells (Grosschedl et al., Cell 38:647-658 (1984); Adams etal., Nature 318:533-538 (1985); Alexander et al., Mol. Cell Biol.7:1436-1444 (1987)), mouse mammary tumor virus control region which isactive in testicular, breast, lymphoid and mast cells (Leder et al.,Cell 45:485-495 (1986)), albumin gene control region which is active inliver (Pinkert et al., Genes and Devel. 1:268-276 (1987)),alpha-fetoprotein gene control region which is active in liver (Krumlaufet al., Mol. Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science235:53-58 1987)), alpha-1 antitrypsin gene control region which isactive in liver (Kelsey et al., Genes and Devel. 1:161-171 (1987)), betaglobin gene control region which is active in myeloid cells (Magram etal., Nature 315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)),myelin basic protein gene control region which is active inoligodendrocyte cells of the brain (Readhead et al., Cell 48:703-712(1987)), myosin light chain-2 gene control region which is active inskeletal muscle (Shani, Nature 314:283-286 (1985)), and gonadotrophicreleasing hormone gene control region which is active in gonadotrophs ofthe hypothalamus (Mason et al., Science 234:1372-1378 (1986)).

In a specific embodiment, a vector is used that contains a promoteroperably linked to nucleic acids encoding a desired protein, or adomain, fragment, derivative or homolog, thereof, one or more origins ofreplication, and optionally, one or more selectable markers (e.g., anantibiotic resistance gene). Exemplary plasmid vectors fortransformation of E. coli cells, include, for example, the pQEexpression vectors (available from Qiagen, Valencia, Calif.; see alsoliterature published by Qiagen describing the system). pQE vectors havea phage T5 promoter (recognized by E. coli RNA polymerase) and a doublelac operator repression module to provide tightly regulated, high-levelexpression of recombinant proteins in E. coli, a synthetic ribosomalbinding site (RBS II) for efficient translation, a 6×His tag codingsequence, t₀ and T1 transcriptional terminators, ColE1 origin ofreplication, and a beta-lactamase gene for conferring ampicillinresistance. The pQE vectors enable placement of a 6×His tag at eitherthe N- or C-terminus of the recombinant protein. Such plasmids includepQE 32, pQE 30, and pQE 31 which provide multiple cloning sites for allthree reading frames and provide for the expression of N-terminally6×His-tagged proteins. Other exemplary plasmid vectors fortransformation of E. coli cells, include, for example, the pETexpression vectors (see, U.S. Pat. No. 4,952,496; available fromNovagen, Madison, Wis.; see, also literature published by Novagendescribing the system). Such plasmids include pET 11a, which containsthe T7lac promoter, T7 terminator, the inducible E. coli lac operator,and the lac repressor gene; pET 12a-c, which contains the T7 promoter,T7 terminator, and the E. coli ompT secretion signal; and pET 15b andpET19b (Novagen, Madison, Wis.), which contain a His-Tag™ leadersequence for use in purification with a His column and a thrombincleavage site that permits cleavage following purification over thecolumn, the T7-lac promoter region and the T7 terminator.

Exemplary of a vector for mammalian cell expression is the HZ24expression vector. The HZ24 expression vector was derived from the pCIvector backbone (Promega). It contains DNA encoding the Beta-lactamaseresistance gene (AmpR), an F1 origin of replication, a Cytomegalovirusimmediate-early enhancer/promoter region (CMV), and an SV40 latepolyadenylation signal (SV40). The expression vector also has aninternal ribosome entry site (IRES) from the ECMV virus (Clontech) andthe mouse dihydrofolate reductase (DHFR) gene.

2. Expression

Hyaluronan binding proteins and hyaluronan-degrading enzymepolypeptides, including soluble hyaluronidase polypeptides, can beproduced by any method known to those of skill in the art including invivo and in vitro methods. Desired proteins can be expressed in anyorganism suitable to produce the required amounts and forms of theproteins, such as for example, the amounts and forms needed foradministration and treatment. Expression hosts include prokaryotic andeukaryotic organisms such as E. coli, yeast, plants, insect cells,mammalian cells, including human cell lines and transgenic animals.Expression hosts can differ in their protein production levels as wellas the types of post-translational modifications that are present on theexpressed proteins. The choice of expression host can be made based onthese and other factors, such as regulatory and safety considerations,production costs and the need and methods for purification.

Many expression vectors are available and known to those of skill in theart and can be used for expression of proteins. The choice of expressionvector will be influenced by the choice of host expression system. Ingeneral, expression vectors can include transcriptional promoters andoptionally enhancers, translational signals, and transcriptional andtranslational termination signals. Expression vectors that are used forstable transformation typically have a selectable marker which allowsselection and maintenance of the transformed cells. In some cases, anorigin of replication can be used to amplify the copy number of thevector.

Hyaluronan binding proteins and hyaluronan-degrading enzymepolypeptides, such as soluble hyaluronidase polypeptides, also can beutilized or expressed as protein fusions. For example, an enzyme fusioncan be generated to add additional functionality to an enzyme. Examplesof enzyme fusion proteins include, but are not limited to, fusions of asignal sequence, a tag such as for localization, e.g. a his₆ tag or amyc tag, or a tag for purification, for example, a GST fusion, and asequence for directing protein secretion and/or membrane association.

a. Prokaryotic Cells

Prokaryotes, especially E. coli, provide a system for producing largeamounts of proteins. Transformation of E. coli is a simple and rapidtechnique well known to those of skill in the art. Expression vectorsfor E. coli can contain inducible promoters, such promoters are usefulfor inducing high levels of protein expression and for expressingproteins that exhibit some toxicity to the host cells. Examples ofinducible promoters include the lac promoter, the trp promoter, thehybrid tac promoter, the T7 and SP6 RNA promoters and the temperatureregulated λPL promoter.

Proteins, such as any provided herein, can be expressed in thecytoplasmic environment of E. coli. The cytoplasm is a reducingenvironment and for some molecules, this can result in the formation ofinsoluble inclusion bodies. Reducing agents such as dithiothreitol andβ-mercaptoethanol and denaturants, such as guanidine-HCl and urea can beused to resolubilize the proteins. An alternative approach is theexpression of proteins in the periplasmic space of bacteria whichprovides an oxidizing environment and chaperonin-like and disulfideisomerases and can lead to the production of soluble protein. Typically,a leader sequence is fused to the protein to be expressed which directsthe protein to the periplasm. The leader is then removed by signalpeptidases inside the periplasm. Examples of periplasmic-targetingleader sequences include the pelB leader from the pectate lyase gene andthe leader derived from the alkaline phosphatase gene. In some cases,periplasmic expression allows leakage of the expressed protein into theculture medium. The secretion of proteins allows quick and simplepurification from the culture supernatant. Proteins that are notsecreted can be obtained from the periplasm by osmotic lysis. Similar tocytoplasmic expression, in some cases proteins can become insoluble anddenaturants and reducing agents can be used to facilitate solubilizationand refolding. Temperature of induction and growth also can influenceexpression levels and solubility, typically temperatures between 25° C.and 37° C. are used. Typically, bacteria produce aglycosylated proteins.Thus, if proteins require glycosylation for function, glycosylation canbe added in vitro after purification from host cells.

b. Yeast Cells

Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe,Yarrowia lipolytica, Kluyveromyces lactis and Pichia pastoris are wellknown yeast expression hosts that can be used for production ofproteins, such as any described herein. Yeast can be transformed withepisomal replicating vectors or by stable chromosomal integration byhomologous recombination. Typically, inducible promoters are used toregulate gene expression. Examples of such promoters include GAL1, GAL7and GAL5 and metallothionein promoters, such as CUP1, AOX1 or otherPichia or other yeast promoter. Expression vectors often include aselectable marker such as LEU2, TRP1, HIS3 and URA3 for selection andmaintenance of the transformed DNA. Proteins expressed in yeast areoften soluble. Co-expression with chaperonins such as Bip and proteindisulfide isomerase can improve expression levels and solubility.Additionally, proteins expressed in yeast can be directed for secretionusing secretion signal peptide fusions such as the yeast mating typealpha-factor secretion signal from Saccharomyces cerevisae and fusionswith yeast cell surface proteins such as the Aga2p mating adhesionreceptor or the Arxula adeninivorans glucoamylase. A protease cleavagesite such as for the Kex-2 protease, can be engineered to remove thefused sequences from the expressed polypeptides as they exit thesecretion pathway. Yeast also is capable of glycosylation atAsn-X-Ser/Thr motifs.

c. Insect Cells

Insect cells, particularly using baculovirus expression, are useful forexpressing polypeptides including the hyaluronan binding proteins andhyaluronan-degrading enzyme polypeptides, such as soluble hyaluronidasepolypeptides. Insect cells express high levels of protein and arecapable of most of the post-translational modifications used by highereukaryotes. Baculovirus have a restrictive host range which improves thesafety and reduces regulatory concerns of eukaryotic expression. Typicalexpression vectors use a promoter for high level expression such as thepolyhedrin promoter of baculovirus. Commonly used baculovirus systemsinclude the baculoviruses such as Autographa californica nuclearpolyhedrosis virus (AcNPV), and the Bombyx mori nuclear polyhedrosisvirus (BmNPV) and an insect cell line such as Sf9 derived fromSpodoptera frugiperda, Pseudaletia unipuncta (A7S) and Danaus plexippus(DpN1). For high-level expression, the nucleotide sequence of themolecule to be expressed is fused immediately downstream of thepolyhedrin initiation codon of the virus. Mammalian secretion signalsare accurately processed in insect cells and can be used to secrete theexpressed protein into the culture medium. In addition, the cell linesPseudaletia unipuncta (A7S) and Danaus plexippus (DpN1) produce proteinswith glycosylation patterns similar to mammalian cell systems.

An alternative expression system in insect cells is the use of stablytransformed cells. Cell lines such as the Schneider 2 (S2) and Kc cells(Drosophila melanogaster) and C7 cells (Aedes albopictus) can be usedfor expression. The Drosophila metallothionein promoter can be used toinduce high levels of expression in the presence of heavy metalinduction with cadmium or copper. Expression vectors are typicallymaintained by the use of selectable markers such as neomycin andhygromycin.

d. Mammalian Cells

Mammalian expression systems can be used to express proteins, includingthe hyaluronan binding proteins and hyaluronan-degrading enzymepolypeptides, such as soluble hyaluronidase polypeptides. Expressionconstructs can be transferred to mammalian cells by viral infection suchas adenovirus or by direct DNA transfer such as liposomes, calciumphosphate, DEAE-dextran and by physical means such as electroporationand microinjection. Expression vectors for mammalian cells typicallyinclude an mRNA cap site, a TATA box, a translational initiationsequence (Kozak consensus sequence) and polyadenylation elements. IRESelements also can be added to permit bicistronic expression with anothergene, such as a selectable marker. Such vectors often includetranscriptional promoter-enhancers for high-level expression, forexample the SV40 promoter-enhancer, the human cytomegalovirus (CMV)promoter and the long terminal repeat of Rous sarcoma virus (RSV). Thesepromoter-enhancers are active in many cell types. Tissue and cell-typepromoters and enhancer regions also can be used for expression.Exemplary promoter/enhancer regions include, but are not limited to,those from genes such as elastase I, insulin, immunoglobulin, mousemammary tumor virus, albumin, alpha fetoprotein, alpha 1 antitrypsin,beta globin, myelin basic protein, myosin light chain 2, andgonadotropic releasing hormone gene control. Selectable markers can beused to select for and maintain cells with the expression construct.Examples of selectable marker genes include, but are not limited to,hygromycin B phosphotransferase, adenosine deaminase, xanthine-guaninephosphoribosyl transferase, aminoglycoside phosphotransferase,dihydrofolate reductase (DHFR) and thymidine kinase. For example,expression can be performed in the presence of methotrexate to selectfor only those cells expressing the DHFR gene. Fusion with cell surfacesignaling molecules such as TCR-ζ and Fc_(ε)RI-γ can direct expressionof the proteins in an active state on the cell surface.

Many cell lines are available for mammalian expression including mouse,rat human, monkey, chicken and hamster cells. Exemplary cell linesinclude but are not limited to CHO, Balb/3T3, HeLa, MT2, mouse NSO(nonsecreting) and other myeloma cell lines, hybridoma andheterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS,NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are availableadapted to serum-free media which facilitates purification of secretedproteins from the cell culture media. Examples include CHO-S cells(Invitrogen, Carlsbad, Calif., cat #11619-012) and the serum free EBNA-1cell line (Pham et al., (2003) Biotechnol. Bioeng. 84:332-342). Celllines also are available that are adapted to grow in special mediaoptimized for maximal expression. For example, DG44 CHO cells areadapted to grow in suspension culture in a chemically defined, animalproduct-free medium.

e. Plants

Transgenic plant cells and plants can be used to express proteins suchas any described herein. Expression constructs are typically transferredto plants using direct DNA transfer such as microprojectile bombardmentand PEG-mediated transfer into protoplasts, and withagrobacterium-mediated transformation. Expression vectors can includepromoter and enhancer sequences, transcriptional termination elementsand translational control elements. Expression vectors andtransformation techniques are usually divided between dicot hosts, suchas Arabidopsis and tobacco, and monocot hosts, such as corn and rice.Examples of plant promoters used for expression include the cauliflowermosaic virus promoter, the nopaline synthetase promoter, the ribosebisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters.Selectable markers such as hygromycin, phosphomannose isomerase andneomycin phosphotransferase are often used to facilitate selection andmaintenance of transformed cells. Transformed plant cells can bemaintained in culture as cells, aggregates (callus tissue) orregenerated into whole plants. Transgenic plant cells also can includealgae engineered to produce hyaluronidase polypeptides. Because plantshave different glycosylation patterns than mammalian cells, this caninfluence the choice of protein produced in these hosts.

3. Purification Techniques

Methods for purification of polypeptides, including hyaluronan bindingproteins and hyaluronan-degrading enzyme polypeptides (e.g. solublehyaluronidase polypeptides) or other proteins, from host cells willdepend on the chosen host cells and expression systems. For secretedmolecules, proteins are generally purified from the culture media afterremoving the cells. For intracellular expression, cells can be lysed andthe proteins purified from the extract. When transgenic organisms suchas transgenic plants and animals are used for expression, tissues ororgans can be used as starting material to make a lysed cell extract.Additionally, transgenic animal production can include the production ofpolypeptides in milk or eggs, which can be collected, and if necessary,the proteins can be extracted and further purified using standardmethods in the art.

Proteins can be purified using standard protein purification techniquesknown in the art including but not limited to, SDS-PAGE, size fractionand size exclusion chromatography, ammonium sulfate precipitation andionic exchange chromatography, such as anion exchange. Affinitypurification techniques also can be utilized to improve the efficiencyand purity of the preparations. For example, antibodies, receptors andother molecules that bind to hyaluronan binding proteins orhyaluronidase enzymes can be used in affinity purification. Expressionconstructs also can be engineered to add an affinity tag to a proteinsuch as a myc epitope, GST fusion or His₆ and affinity purified with mycantibody, glutathione resin and Ni-resin, respectively. Purity can beassessed by any method known in the art including gel electrophoresisand staining and spectrophotometric techniques. Purified rHuPH20compositions, as provided herein, typically have a specific activity ofat least 70,000 to 100,000 Units/mg, for example, about 120,000Units/mg. The specific activity can vary upon modification, such as witha polymer.

4. PEGylation of Hyaluronan-Degrading Enzyme Polypeptides

Polyethylene glycol (PEG) has been widely used in biomaterials,biotechnology and medicine primarily because PEG is a biocompatible,nontoxic, water-soluble polymer that is typically nonimmunogenic (Zhaoand Harris, ACS Symposium Series 680: 458-72, 1997). In the area of drugdelivery, PEG derivatives have been widely used in covalent attachment(i. e., “PEGylation”) to proteins to reduce immunogenicity, proteolysisand kidney clearance and to enhance solubility (Zalipsky, Adv. Drug Del.Rev. 16:157-82, 1995). Similarly, PEG has been attached to low molecularweight, relatively hydrophobic drugs to enhance solubility, reducetoxicity and alter biodistribution. Typically, PEGylated drugs areinjected as solutions.

A closely related application is synthesis of crosslinked degradable PEGnetworks or formulations for use in drug delivery since much of the samechemistry used in design of degradable, soluble drug carriers can alsobe used in design of degradable gels (Sawhney et al., Macromolecules 26:581-87, 1993). It also is known that intermacromolecular complexes canbe formed by mixing solutions of two complementary polymers. Suchcomplexes are generally stabilized by electrostatic interactions(polyanion-polycation) and/or hydrogen bonds (polyacid-polybase) betweenthe polymers involved, and/or by hydrophobic interactions between thepolymers in an aqueous surrounding (Krupers et al., Eur. Polym J.32:785-790, 1996). For example, mixing solutions of polyacrylic acid(PAAc) and polyethylene oxide (PEO) under the proper conditions resultsin the formation of complexes based mostly on hydrogen bonding.Dissociation of these complexes at physiologic conditions has been usedfor delivery of free drugs (i.e., non-PEGylated). In addition, complexesof complementary polymers have been formed from both homopolymers andcopolymers.

Numerous reagents for PEGylation have been described in the art. Suchreagents include, but are not limited to, N-hydroxysuccinimidyl (NHS)activated PEG, succinimidyl mPEG, mPEG₂-N-hydroxysuccinimide, mPEGsuccinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEGsuccinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acidsuccinimidyl ester, homobifunctional PEG-succinimidyl propionate,homobifunctional PEG propionaldehyde, homobifunctional PEGbutyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonatePEG, mPEG-benzotriazole carbonate, propionaldehyde PEG, mPEGbutyraldehyde, branched mPEG₂ butyraldehyde, mPEG acetyl, mPEGpiperidone, mPEG methylketone, mPEG “linkerless” maleimide, mPEG vinylsulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyldisulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylatePEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see e.g., Monfardiniet al., Bioconjugate Chem. 6:62-69, 1995; Veronese et al., J. BioactiveCompatible Polymers 12:197-207, 1997; U.S. Pat. No. 5,672,662; U.S. Pat.No. 5,932,462; U.S. Pat. No. 6,495,659; U.S. Pat. No. 6,737,505; U.S.Pat. No. 4,002,531; U.S. Pat. No. 4,179,337; U.S. Pat. No. 5,122,614;U.S. 5,324, 844; U.S. Pat. No. 5,446,090; U.S. Pat. No. 5,612,460; U.S.Pat. No. 5,643,575; U.S. Pat. No. 5,766,581; U.S. 5,795, 569; U.S. Pat.No. 5,808,096; U.S. Pat. No. 5,900,461; U.S. Pat. No. 5,919,455; U.S.Pat. No. 5,985,263; U.S. 5,990, 237; U.S. Pat. No. 6,113,906; U.S. Pat.No. 6,214,966; U.S. Pat. No. 6,258,351; U.S. Pat. No. 6,340,742; U.S.Pat. No. 6,413,507; U.S. Pat. No. 6,420,339; U.S. Pat. No. 6,437,025;U.S. Pat. No. 6,448,369; U.S. Pat. No. 6,461,802; U.S. Pat. No.6,828,401; U.S. Pat. No. 6,858,736; U.S. 2001/0021763; U.S.2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430; U.S. 2002/0072573;U.S. 2002/0156047; U.S. 2003/0114647; U.S. 2003/0143596; U.S.2003/0158333; U.S. 2003/0220447; U.S. 2004/0013637; US 2004/0235734;WO05000360; U.S. 2005/0114037; U.S. 2005/0171328; U.S. 2005/0209416; EP1064951; EP 0822199; WO 01076640; WO 0002017; WO 0249673; WO 9428024;and WO 0187925).

In one example, the polyethylene glycol has a molecular weight rangingfrom about 3 kD to about 50 kD, and typically from about 5 kD to about30 kD. Covalent attachment of the PEG to the drug (known as“PEGylation”) can be accomplished by known chemical synthesistechniques. For example, the PEGylation of protein can be accomplishedby reacting NHS-activated PEG with the protein under suitable reactionconditions.

While numerous reactions have been described for PEGylation, those thatare most generally applicable confer directionality, utilize mildreaction conditions, and do not necessitate extensive downstreamprocessing to remove toxic catalysts or bi-products. For instance,monomethoxy PEG (mPEG) has only one reactive terminal hydroxyl, and thusits use limits some of the heterogeneity of the resulting PEG-proteinproduct mixture. Activation of the hydroxyl group at the end of thepolymer opposite to the terminal methoxy group is generally necessary toaccomplish efficient protein PEGylation, with the aim being to make thederivatised PEG more susceptible to nucleophilic attack. The attackingnucleophile is usually the epsilon-amino group of a lysyl residue, butother amines also can react (e.g. the N-terminal alpha-amine or the ringamines of histidine) if local conditions are favorable. A more directedattachment is possible in proteins containing a single lysine orcysteine. The latter residue can be targeted by PEG-maleimide forthiol-specific modification. Alternatively, PEG hydrazide can be reactedwith a periodate oxidized hyaluronan-degrading enzyme and reduced in thepresence of NaCNBH₃. More specifically, PEGylated CMP sugars can bereacted with a hyaluronan-degrading enzyme in the presence ofappropriate glycosyl-transferases. One technique is the “PEGylation”technique where a number of polymeric molecules are coupled to thepolypeptide in question. When using this technique the immune system hasdifficulties in recognizing the epitopes on the polypeptide's surfaceresponsible for the formation of antibodies, thereby reducing the immuneresponse. For polypeptides introduced directly into the circulatorysystem of the human body to give a particular physiological effect (i.e.pharmaceuticals) the typical potential immune response is an IgG and/orIgM response, while polypeptides which are inhaled through therespiratory system (i.e. industrial polypeptide) potentially can causean IgE response (i.e. allergic response). One of the theories explainingthe reduced immune response is that the polymeric molecule(s) shield(s)epitope(s) on the surface of the polypeptide responsible for the immuneresponse leading to antibody formation. Another theory or at least apartial factor is that the heavier the conjugate is, the more reducedimmune response is obtained.

Typically, to make the PEGylated hyaluronan-degrading enzymes providedherein, including the PEGylated hyaluronidases, PEG moieties areconjugated, via covalent attachment, to the polypeptides. Techniques forPEGylation include, but are not limited to, specialized linkers andcoupling chemistries (see e.g., Roberts, Adv. Drug Deliv. Rev.54:459-476, 2002), attachment of multiple PEG moieties to a singleconjugation site (such as via use of branched PEGs; see e.g., Guiotto etal., Bioorg. Med. Chem. Lett. 12:177-180, 2002), site-specificPEGylation and/or mono-PEGylation (see e.g., Chapman et al., NatureBiotech. 17:780-783, 1999), and site-directed enzymatic PEGylation (seee.g., Sato, Adv. Drug Deliv. Rev., 54:487-504, 2002). Methods andtechniques described in the art can produce proteins having 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more than 10 PEG or PEG derivatives attached to asingle protein molecule (see e.g., U.S. 2006/0104968).

As an exemplary illustrative method for making PEGylatedhyaluronan-degrading enzymes, such as PEGylated hyaluronidases, PEGaldehydes, succinimides and carbonates have each been applied toconjugate PEG moieties, typically succinimidyl PEGs, to rHuPH20. Forexample, rHuPH20 has been conjugated with exemplary succinimidyl monoPEG(mPEG) reagents including mPEG-Succinimidyl Propionates (mPEG-SPA),mPEG-Succinimidyl Butanoates (mPEG-SBA), and (for attaching “branched”PEGs) mPEG2-N-Hydroxylsuccinimide. These PEGylated succinimidyl esterscontain different length carbon backbones between the PEG group and theactivated cross-linker, and either a single or branched PEG group. Thesedifferences can be used, for example, to provide for different reactionkinetics and to potentially restrict sites available for PEG attachmentto rHuPH20 during the conjugation process.

Succinimidyl PEGs (as above) comprising either linear or branched PEGscan be conjugated to rHuPH20. PEGs can used to generate rHuPH20sreproducibly containing molecules having, on the average, between aboutthree to six or three to six PEG molecules per hyaluronidase. SuchPEGylated rHuPH20 compositions can be readily purified to yieldcompositions having specific activities of approximately 25,000 or30,000 Unit/mg protein hyaluronidase activity, and being substantiallyfree of non-PEGylated rHuPH20 (less than 5% non-PEGylated).

Using various PEG reagents, exemplary versions of hyaluronan-degradingenzymes, in particular soluble human recombinant hyaluronidases (e.g.rHuPH20), can be prepared, for example, using mPEG-SBA (30 kD), mPEG-SMB(30 kD), and branched versions based on mPEG2-NHS (40 kD) and mPEG2-NHS(60 kD). PEGylated versions of rHuPH20 have been generated using NHSchemistries, as well as carbonates, and aldehydes, using each of thefollowing reagents: mPEG2-NHS-40K branched, mPEG-NHS-10K branched,mPEG-NHS-20K branched, mPEG2-NHS-60K branched; mPEG-SBA-5K,mPEG-SBA-20K, mPEG-SBA-30K; mPEG-SMB-20K, mPEG-SMB-30K;mPEG-butyrldehyde; mPEG-SPA-20K, mPEG-SPA-30K; and PEG-NHS-5K-biotin.PEGylated hyaluronidases have also been prepared using PEG reagentsavailable from Dowpharma, a division of Dow Chemical Corporation;including hyaluronidases PEGylated with Dowpharma'sp-nitrophenyl-carbonate PEG (30 kDa) and with propionaldehyde PEG (30kDa).

In one example, the PEGylation includes conjugation of mPEG-SBA, forexample, mPEG-SBA-30K (having a molecular weight of about 30 kDa) oranother succinimidyl esters of PEG butanoic acid derivative, to asoluble hyaluronidase. Succinimidyl esters of PEG butanoic acidderivatives, such as mPEG-SBA-30K readily couple to amino groups ofproteins. For example, covalent conjugation of m-PEG-SBA-30K and rHuPH20(which is approximately 60 KDa in size) provides stable amide bondsbetween rHuPH20 and mPEG, as shown in Scheme 1, below.

Typically, the mPEG-SBA-30K or other PEG is added to thehyaluronan-degrading enzyme, in some instances a hyaluronidase, at aPEG:polypeptide molar ratio of 10:1 in a suitable buffer, e.g. 130 mMNaCl/10 mM HEPES at pH 6.8 or 70 mM phosphate buffer, pH 7, followed bysterilization, e.g. sterile filtration, and continued conjugation, forexample, with stirring, overnight at 4° C. in a cold room. In oneexample, the conjugated PEG-hyaluronan-degrading enzyme is concentratedand buffer-exchanged.

Other methods of coupling succinimidyl esters of PEG butanoic acidderivatives, such as mPEG-SBA-30K are known in the art (see e.g., U.S.Pat. No. 5,672,662; U.S. Pat. No. 6,737,505; and U.S. 2004/0235734). Forexample, a polypeptide, such as a hyaluronan-degrading enzyme (e.g. ahyaluronidase), can be coupled to an NHS activated PEG derivative byreaction in a borate buffer (0.1 M, pH 8.0) for one hour at 4° C. Theresulting PEGylated protein can be purified by ultrafiltration.Alternatively, PEGylation of a bovine alkaline phosphatase can beaccomplished by mixing the phosphatase with mPEG-SBA in a buffercontaining 0.2 M sodium phosphate and 0.5 M NaCl (pH 7.5) at 4° C. for30 minutes. Unreacted PEG can be removed by ultrafiltration. Anothermethod reacts polypeptide with mPEG-SBA in deionized water to whichtriethylamine is added to raise the pH to 7.2-9. The resulting mixtureis stirred at room temperature for several hours to complete thePEGylation.

Methods for PEGylation of hyaluronan-degrading polypeptides, including,for example, animal-derived hyaluronidases and bacterialhyaluronan-degrading enzymes, are known to one of skill in the art. See,for example, European Patent No. EP 0400472, which describes thePEGylation of bovine testes hyaluorindase and chondroitin ABC lyase.Also, U.S. Publication No. 2006014968 describes PEGylation of a humanhyaluronidase derived from human PH20. For example, the PEGylatedhyaluronan-degrading enzyme generally contains at least 3 PEG moietiesper molecule. For example, the hyaluronan-degrading enzyme can have aPEG to protein molar ratio between 5:1 and 9:1, for example, 7:1.

G. METHODS OF ASSESSING ACTIVITY AND MONITORING EFFECTS OFANTI-HYALURONAN AGENTS

Anti-hyaluronan agents, for example hyaluronan-degrading enzymes, suchas a hyaluronidase or modified hyaluronidase (e.g. PH20 or PEGPH20) actas therapeutic agents either alone, or in combination with secondaryagents such as chemotherapeutic drugs, for the treatment ofhyaluronan-associated diseases and conditions, in particular cancers(see for example, US 2010/0003238 and WO09/128917). In addition, asdescribed elsewhere herein, therapy with an anti-hyaluronan agent, forexample a hyaluronan-degrading enzyme, can be accompanied by treatmentwith a corticosteroid to minimize the systemic, for examplemusculoskeletal, side effects of the PEGylated hyaluronidase. The HABPcompanion diagnostics, such as TSG-6-LM or TSG-6-LM:Fc or variantsthereof, provided herein can be used in conjunction with ananti-hyaluronan agent therapy, for example a hyaluronan-degrading enzymetherapy, used for the treatment of hyaluronan-associated diseases ordisorders, such as cancer, in order to monitor responsiveness andefficacy of treatment with the agent (e.g. a hyaluronan-degradingenzyme). In addition, adjunct or supplementary methods also can beutilized to assess the effects of anti-hyaluronan agents (e.g.hyaluronan-degrading enzymes) in treatment alone or in combination withcorticosteroids. It is within the level of one of skill in the art toassess amelioration of side effects by corticosteroids, as wells asefficacy, tolerability and pharmacokinetic studies of anti-hyaluronanagent therapy, including hyaluronan-degrading enzyme therapy. Thissection provides description of adjunct or supplementary methods thatcan be used to assess efficacy, responsiveness, tolerability and/orpharmacokinetics of a hyaluronan-degrading enzyme therapy.

1. Methods to Assess Side Effects

In vivo assays can be used to assess the efficacy of corticosteroids onthe amelioration or elimination of the musculoskeletal side effects thatcan be caused by an anti-hyaluronan agents, for example ahyaluronan-degrading enzyme, such as a hyaluronidase or hyaluronidasemodified to exhibit increased systemic half-life (e.g. PH20 or PEGPH20).Side effects that can be assessed include, for example, muscle and jointpain, stiffness of upper and lower extremities, cramping, myositis,muscle soreness and tenderness over the entire body, weakness, fatigueand/or a decrease in range of motion at knee and elbow joints. Assays toassess side effects can include animal models wherein the animal can beobserved for reduced movement, behavior or posture changes, radiographicfindings, histopathological changes and other notable clinicalobservations. Other assays can include clinical trials in human subjectswherein patients can be questioned regarding symptoms, assessed byphysical examination, imaging (for example by MRI or PET) or byradiologic evaluation. Amelioration of a side effect caused byadministration of an anti-hyaluronan agent (e.g. a hyaluronan-degradingenzyme agent) is observed when the side effect is ameliorated,eliminated, lessened or reduced in the presence of the corticosteroidcompared to in its absence.

In such examples, the dose of anti-hyaluronan agent (e.g. ahyaluronan-degrading enzyme) and/or corticosteroid can be varied toidentify the optimal or minimal dose required to achieve activity whileameliorating side effects. Such studies are within the level of one ofskill in the art. Further, the dosage regime can be varied. For example,studies can be performed using a dosage schedule of hyaluronan-degradingenzyme monthly, biweekly, once a week, twice a week, three times a week,four times a week or more. Further, the corticosteroid can beadministered prior to, concurrently and/or subsequent to administrationof the anti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme).

For example, in vivo animal models can be utilized to assess the abilityof corticosteroids, such as dexamethasone, to ameliorate or eliminatethe side effects associated with anti-hyaluronan agent (e.g.hyaluronan-degrading enzyme) administration. Animal models can includenon-human primates such as cynomolgus monkeys or rhesus macaques, dogs,for example beagle dogs, or any other animal that exhibits adverse sideeffects in response to treatment with an anti-Hyaluronan agent (e.g. ahyaluronan-degrading enzyme, such as a PEGylated hyaluronidase, forexample PEGPH20) treatment. The animal models can be dosed with ananti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme) in thepresence or absence of corticosteroid and musculoskeletal effectsobserved or measured.

For example, animals such as cynomolgus monkeys, beagles or othersimilar animal model capable of observable or measurable musculoskeletalevents can be treated with hyaluronan-degrading enzyme in the presenceor absence of corticosteroid. In one example, a group of animals, forexample cynomolgus monkeys or beagles, is administered with ananti-hyaluronan agent, such as a hyaluronan-degrading enzyme alone, forexample a PEGylated hyaluronidase, such as by intravenousadministration. For example, administration can be twice weekly.Treatment can continue until changes in limb joint range-of-motion areobserved at the knee and elbow joints or stiffness or reduced mobilityis observed. Then, another group of animals can be treated with theanti-hyaluronan agent (e.g. hyaluronan-degrading enzyme) andcorticosteroid administered, such as by oral doses of dexamethasone orother corticosteroid, given on the same day as the anti-hyaluronan agent(e.g. hyaluronan-degrading enzyme) administration. The groups of animalscan then be compared for example, via physical examination of jointrange-of-motion or other reduced mobility, histopathology of the joints,palpation for stiffness, or imaging known to those of skill in the art,to assess the ability of the corticosteroid, such as dexamethasone, toameliorate the anti-hyaluronan agent-mediated, such ashyaluronan-degrading enzyme-mediated, musculoskeletal side effects.Dose, dosing frequency, route of administration, and timing of dosing ofcorticosteroid, such as dexamethasone, can be varied to optimize theeffectiveness of the corticosteroid.

In another example, the efficacy of corticosteroids such asdexamethasone on the amelioration or elimination of the adverse sideeffects associated with an anti-hyaluronan agent (e.g. ahyaluronan-degrading enzyme) administration can be assessed in humanpatients with solid tumors. For example human patients can be dosed toexamine the ability of corticosteroid to ameliorate and/or eliminateanti-hyaluronan agent-mediated (e.g. hyaluronan-degradingenzyme-mediated) adverse events including, but not limited to any one ormore of the following: muscle and joint pain/stiffness of upper andlower extremities, cramping, muscle, myositis muscle soreness andtenderness over the entire body, weakness and fatigue. Patients can betreated with an anti-hyaluronan agent (e.g. a hyaluronan-degradingenzyme) with or without co-treatment with a corticosteroid such asdexamethasone. During and after administration of an anti-hyaluronanagent (e.g. a hyaluronan-degrading enzyme), side effects of bothtreatment groups can be assessed. A physician can determine the severityof the symptoms by physical examination of the subject including forexample, patient complaints, vital signs, changes in body weight,12-lead ECG, echocardiogram, clinical chemistry, or imaging (MRI, PET orradiologic evaluation). The severity of symptoms can then be quantifiedusing the NCI Common Terminology Criteria for Adverse Events (CTCAE)grading system. The CTCAE is a descriptive terminology utilized forAdverse Event (AE) reporting. A grading (severity) scale is provided foreach AE term. The CTCAE displays Grades 1 through 5, with clinicaldescriptions for severity for each adverse event based on the followinggeneral guideline: Grade 1 (Mild AE); Grade 2 (Moderate AE); Grade 3(Severe AE); Grade 4 (Life-threatening or disabling AE); and Grade 5(Death related to AE). The ability of a corticosteroid to ameliorateadverse side effects associated with administration of ananti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme) can bemeasured by the observation of a reduction in grading or severity on theCTCAE scale in one or more adverse side effects in subjects treated withthe anti-hyaluronan agent (e.g. hyaluronan-degrading enzyme) andcorticosteroid as compared to subjects treated with the sameanti-hyaluronan agent (e.g. hyaluronan-degrading enzyme) alone, i.e.,the severity of the side effects, is reduced from Grade 3 to Grade 1 orGrade 2.

In another example, human patients can be dosed to assess tolerabilityby escalating the dose of an anti-hyaluronan agent (e.g. ahyaluronan-degrading enzyme) and assessing the dose-limiting toxicity asmeasured by severity of side effects. In such an example, a maximumtolerated dose of an anti-hyaluronan agent (e.g. a hyaluronan-degradingenzyme) that can be tolerated in the presence of an ameliorating agentsuch as a corticosteroid can be determined. Treatment regimens caninclude a dose escalation wherein each patient receives a higher dose ofhyaluronan-degrading enzyme at the same dose level of corticosteroid.Patients can be monitored for adverse events to determine the highestdose of hyaluronan-degrading enzyme that can be administered with acorticosteroid before side effects are no longer tolerated. Tolerabilitycan be measured based on the severity of symptoms emerging during andafter treatment. Doses of an anti-hyaluronan agent (e.g. ahyaluronan-degrading enzyme) can be escalated until adverse effectsreach a predetermined level, for example, Grade 3. Dosing regimens canalso include a tapering of the amount of corticosteroid administered toexamine the continued need for corticosteroid and the possibility ofacclimation to the anti-hyaluronan agent with respect to resulting sideeffects.

2. Evaluating Biomarkers Associated with Activity of an Anti-HyaluronanAgent (e.g. a Hyaluronan-Degrading Enzyme Activity)

As described herein, the extent and level of HA phenotypes is abiomarker that is associated with and correlates to efficacy andactivity of an anti-hyaluronan agent (e.g. hyaluronan-degrading enzyme).For example, for cancer patients with tumors such as advanced solidtumors, reduced tumor- and stroma-associated is a biomarker of activityof an administered hyaluronan-degrading enzyme. An HABP binding assaysto detect HA present in tissue (e.g. tumor biopsy) or bodily fluids(e.g. plasma) as described elsewhere herein can be performed to evaluateand monitor the therapeutic effect of an anti-hyaluronan (e.g.hyaluronan-degrading enzyme).

In addition, assays can be performed separately or in conjugation withHABP assays described herein used to detect HA in tissue (e.g. tumorbiopsy) or bodily fluids (e.g. plasma) to further assess the effects ofan anti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme) onhyaluronan inhibition or degradation activity. In particular, fortreatment of a hyaluronan-associated disease or condition, such ascancer, clinical measures or biomarkers associated with activity of ananti-Hyaluronan agent, for example a hyaluronan-degrading enzymeactivity include, but are not limited to, reduced tumor metabolicactivity, increased apparent diffusion and enhanced tumor perfusionand/or increase in HA catabolites. Additional assays to measure suchbiomarkers can include, but are not limited to, measurements ofhyaluronan catabolites in blood or urine, measurements of hyaluronidaseactivity in plasma, or measurements of interstitial fluid pressure,vascular volume or water content in tumors. It is within the level ofone skilled in the art to perform such assays.

These assays can be performed in animal models treated with ahyaluronan-degrading enzyme or in human patients. For example, animalmodels of hyaluronan-associated diseases, disorders or conditions can beutilized to assess the in vivo affect of administration of ananti-hyaluronan agent, for example a hyaluronan-degrading enzyme, suchas a hyaluronidase or hyaluronidase modified to exhibit increasedhalf-life (e.g. PH20 or PEGPH20). Another agent, such as achemotherapeutic agent can also be included in the assessment ofactivity. Exemplary hyaluronan-associated diseases for which anappropriate animal model can be utilized include solid tumors, forexample, late-stage cancers, a metastatic cancers, undifferentiatedcancers, ovarian cancer, in situ carcinoma (ISC), squamous cellcarcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lungcancer, breast cancer, colon cancer and other cancers. Also exemplary ofhyaluronan-associated diseases and disorders are inflammatory diseases,disc pressure, cancer and edema, for example, edema caused by organtransplant, stroke, brain trauma or other injury.

Animal models can include, but are not limited to, mice, rats, rabbits,dogs, guinea pigs and non-human primate models, such as cynomolgusmonkeys or rhesus macaques. In some examples, immunodeficient mice, suchas nude mice or SCID mice, are transplanted with a tumor cell line froma hyaluronan-associated cancer to establish an animal model of thatcancer. Exemplary cell lines from hyaluronan-associated cancers include,but are not limited to, PC3 prostate carcinoma cells, BxPC-3 pancreaticadenocarcinoma cells, MDA-MB-231 breast carcinoma cells, MCF-7 breasttumor cells, BT474 breast tumor cells, Tramp C2 prostate tumor cells andMat-LyLu prostate cancer cells, and other cell lines described hereinthat are hyaluronan associated, e.g. contain elevated levels ofhyaluronan. An anti-hyaluronan agent, such as a hyaluronan-degradingenzyme, can then be administered to the animal with or without acorticosteroid such as dexamethasone, to assess the effects of thecorticosteroid on anti-hyaluronan activity by measuring, for example,hyaluronan levels or content. Hyaluronan content can be measured bystaining tumor tissue samples for hyaluronan or by measuring solublehyaluronan levels in plasma. Other measurements of anti-hyaluronanactivity include the assessment of tumor volume, formation or size ofhalos, interstitial fluid pressure, water content and/or vascularvolume.

In other examples, dogs such as beagle dogs, can be treated with ananti-hyaluronan agent in the presence or absence of a corticosteroid,such as dexamethasone. Tissues such as skin or skeletal muscle tissueare biopsied and stained for hyaluronan and evaluated visually. Tissuesfrom animals treated with an anti-hyaluronan agent alone are thencompared to tissues from aminals treated with the anti-hyaluronan agentand corticosteroid to measure the effect of the corticosteroid onanti-hyaluronan activity.

Assays for activity of an anti-hyaluronan agent, such as ahyaluronan-degrading enzyme activity, also can be performed in humansubjects. For example, assays to measure a biomarker associated with ananti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme) activity canbe performed on human subjects known or suspected of having ahyaluronan-associated disease or condition (e.g. cancer) and that havebeen treated with a hyaluronan-degrading enzyme (e.g. PEGPH20).

a. Assays to Assess the Activity of a Hyaluronan Degrading Enzyme

The activity of a hyaluronan degrading enzyme can be assessed usingmethods well known in the art. For example, the USP XXII assay forhyaluronidase determines activity indirectly by measuring the amount ofundegraded hyaluronic acid, or hyaluronan, (HA) substrate remainingafter the enzyme is allowed to react with the HA for 30 min at 37° C.(USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention,Inc, Rockville, Md.). A Hyaluronidase Reference Standard (USP) orNational Formulary (NF) Standard Hyaluronidase solution can be used inan assay to ascertain the activity, in units, of any hyaluronidase. Inone example, activity is measured using a microturbidity assay. This isbased on the formation of an insoluble precipitate when hyaluronic acidbinds with serum albumin. The activity is measured by incubatinghyaluronidase or a sample containing hyaluronidase, for example blood orplasma, with sodium hyaluronate (hyaluronic acid) for a set period oftime (e.g. 10 minutes) and then precipitating the undigested sodiumhyaluronate with the addition of acidified serum albumin. The turbidityof the resulting sample is measured at 640 nm after an additionaldevelopment period. The decrease in turbidity resulting fromhyaluronidase activity on the sodium hyaluronate substrate is a measureof hyaluronidase enzymatic activity.

In another example, hyaluronidase activity is measured using amicrotiter assay in which residual biotinylated hyaluronic acid ismeasured following incubation with hyaluronidase or a sample containinghyaluronidase, for example, blood or plasma (see e.g. Frost and Stern(1997) Anal. Biochem. 251:263-269, U.S. Patent Publication No.20050260186). The free carboxyl groups on the glucuronic acid residuesof hyaluronic acid are biotinylated, and the biotinylated hyaluronicacid substrate is covalently coupled to a microtiter plate. Followingincubation with hyaluronidase, the residual biotinylated hyaluronic acidsubstrate is detected using an avidin-peroxidase reaction, and comparedto that obtained following reaction with hyaluronidase standards ofknown activity. Other assays to measure hyaluronidase activity also areknown in the art and can be used in the methods herein (see e.g. Delpechet al., (1995) Anal. Biochem. 229:35-41; Takahashi et al., (2003) Anal.Biochem. 322:257-263).

The ability of an active hyaluronan degrading enzyme, such as a modifiedsoluble hyaluronidase (eg PEGylated PH20) to act as a spreading ordiffusing agent, e.g. for chemotherapeutics, also can be assessed. Forexample, trypan blue dye can be injected, such as subcutaneously orintradermally, with or without a hyaluronan degrading enzyme into thelateral skin on each side of nude mice. The dye area is then measured,such as with a microcaliper, to determine the ability of the hyaluronandegrading enzyme to act as a spreading agent (see e.g. U.S. PublishedPatent No. 20060104968).

b. Measurement of HA Catabolites

In another example, blood and urine can be collected at different timepoints throughout patient treatment and assayed for catabolites ofhyaluronan. The presence of catabolites is indicative of the degradationof hyaluronan and is thus a measure of the activity of hyaluronidase.Plasma enzyme also can be assessed and measured over time followingadministration. For example, HA catabolites, which are HA-disaccharidebreakdown products, can be assessed using high-performance liquidchromatography (HPLC) to separate and measure saccharide peak areas.Example 15 exemplifies this assay.

c. Tumor Metabolic Activity

A reduction in tumor metabolic activity is associated withanti-hyaluronan agent (e.g. hyaluronan-degrading enzyme) activity. Tumormetabolic activity can be assessed using standard procedures known inthe art. For example, [18F]-fluorodeoxyglucose positron emissiontomography (FDG-PET) can be used. PET is a non-invasive diagnostic thatprovides images and quantitative parameters of perfusion, cellviability, proliferation and/or metabolic activity of tissues. Theimages result from the use of different biological substances (e.g.sugars, amino acids, metabolic precursors, hormones) labelled withpositron emitting radioisotopes. For example, FDG is an analogue ofglucose and is taken up by living cells via the first stages of normalglucose pathway. In cancers, increased glycolytic activity existsresulting in trapping of FDG in the cancer cell. A decrease in FDGtrapping correlates with a decreased tumor metabolic activity andanti-tumorigenic activity. Guidelines for PET imaging are known to oneof skill in the art and should be followed by any treating physician ortechnician.

d. Increased Apparent Diffusion and Enhanced Tumor Perfusion

Additional methods of assessing anti-hyaluronan agent (e.g.hyaluronan-degrading enzyme) activity include assays that assess thediffusion of water in tissues. As discussed elsewhere herein, tissuesthat accumulate hyaluronan generally have a higher interstitial fluidpressure than normal tissue due to the concomitant accumulation ofwater. Thus, tissues that accumulate HA, such as tumors, have highinterstitial fluid pressure, which can be measured by various methodsknown in the art. For example, diffusion MRI, such as ADC MRI or DCEMRI, can be used. Diffusion of water can be assessed by theseprocedures, and is directly correlated to presence of hyaluronan-richtissues, such as solid tumors (see e.g. Chenevert et al. (1997) ClinicalCancer Research, 3:1457-1466). For example, tumors that accumulatehyaluronan have a distinguishable increase in ADC MRI or DCE MRI becauseof increased perfusion. Such assays can be performed in the presence andabsence of a hyaluronan-degrading enzyme, and results compared. Methodsof measuring diffusion are a useful measure of assessing cellularchanges following such therapies.

3. Tumor Size and Volume

Activity of an anti-hyaluronan agent (e.g. hyaluronan-degrading enzymes)is associated with reductions in tumor size and/or volume. Tumor sizeand volume can be monitored based on techniques known to one of skill inthe art. For example, tumor size and volume can be monitored byradiography, ultrasound imaging, necropsy, by use of calipers, bymicroCT or by ¹⁸F-FDG-PET. Tumor size also can be assessed visually. Inparticular examples, tumor size (diameter) is measured directly usingcalipers.

In other examples, tumor volume can be measured using an average ofmeasurements of tumor diameter (D) obtained by caliper or ultrasoundassessments. For example, tumor volume can be determined usingVisualSonics Vevo 770 high-resolution ultrasound or other similarultrasound. The volume can be determined from the formula V=D³×π/6 (fordiameter measured using calipers) or V=D²×d×π/6 (for diameter measuredusing ultrasound where d is the depth or thickness). For example,caliper measurements can be made of the tumor length (l) and width (w)and tumor volume calculated as length×width×0.52. In another example,microCT scans can be used to measure tumor volume (see e.g. Huang et al.(2009) PNAS, 106:3426-3430). As an example, mice can be injected withOptiray Pharmacy ioversol injection 74% contrast medium (e.g. 741 mg ofioversol/mL), mice anesthetized, and CT scanning done using a MicroCat1A scanner or other similar scanner (e.g. IMTek) (40 kV, 600 μA, 196rotation steps, total angle or rotation=196). The images can bereconstructed using software (e.g. RVA3 software program; ImTek). Tumorvolumes can be determined by using available software (e.g. Amira 3.1software; Mercury Computer Systems). Tumor volume or size also can bedetermined based on size or weight of a tumor.

The percent of tumor growth inhibition can be calculated based on thevolume using the equation: % TGI=[1−(T_(n)−T₀)÷(C_(n)−C₀)]×100%, where“T_(n)” is the average tumor volume for the treatment group at day “n”after the final dose of hyaluronan-degrading enzyme; “T₀” is the averagetumor volume in that treatment group at day 0, before treatment; “C_(n)”is the average tumor volume for the corresponding control group at day“n”; and “C₀” is the average tumor volume in the control group at day 0,before treatment. Statistical analysis of tumor volumes can bedetermined.

4. Pharmacokinetic and Pharmacodynamic Assays

Pharmacokinetic or pharmacodynamic studies can be performed using animalmodels or can be performed during studies with patients to assess thepharmacokinetic properties of an anti-hyaluronan agent, for example ahyaluronan degrading enzyme, such as a hyaluronidase or modifiedhyaluronidase (e.g. PEGPH20). Animal models include, but are not limitedto, mice, rats, rabbits, dogs, guinea pigs and non-human primate models,such as cynomolgus monkeys or rhesus macaques. In some instances,pharmacokinetic or pharmacodynamic studies are performed using healthyanimals. In other examples, the studies are performed using animalmodels of a disease for which therapy with hyaluronan is considered,such as animal models of any hyaluronan-associated disease or disorder,for example a tumor model.

The pharmacokinetic properties of an anti-hyaluronan agent (e.g. ahyaluronan-degrading enzyme, such as a modified hyaluronidase) can beassessed by measuring such parameters as the maximum (peak)concentration (C_(max)), the peak time (i.e. when maximum concentrationoccurs; T_(max)), the minimum concentration (i.e. the minimumconcentration between doses; C_(min)), the elimination half-life(T_(1/2)) and area under the curve (i.e. the area under the curvegenerated by plotting time versus concentration; AUC), followingadministration. The absolute bioavailability of the agent or enzyme(e.g. a hyaluronidase) can be determined by comparing the area under thecurve following subcutaneous delivery (AUC_(sc)) with the AUC followingintravenous delivery (AUC_(iv)). Absolute bioavailability (F), can becalculated using the formula:F=([AUC]_(sc)×dose_(sc))/([AUC]_(iv)×dose_(iv)). A range of doses anddifferent dosing frequency of dosing can be administered in thepharmacokinetic studies to assess the effect of increasing or decreasingconcentrations of an anti-Hyaluronan agent, for example ahyaluronan-degrading enzyme, such as a hyaluronidase or modifiedhyaluronidase (e.g. PEGylated PH20) in the dose.

H. KITS AND ARTICLES OF MANUFACTURE

Provided herein are kits for use in selecting patients for treatmentwith an anti-hyaluronan agent (e.g. a hyaluronan degrading enzyme), forpredicting the efficacy of treatment with an anti-hyaluronan agent (e.g.hyaluronan degrading enzyme), for determining the prognosis of a patientwith an HA-associated diseases, or for monitoring the efficacy oftreatment with an anti-hyaluronan agent (e.g. a hyaluronan degradingenzyme) for the treatment of HA-associated diseases, in particularcancer. The kits provided herein contain an HABP reagent provided hereinfor the detection and quantitation of hyaluronan in a sample andoptionally, reagents for performing the methods. For example, kits canadditionally contain reagents for collection of tissues, preparation andprocessing of tissues, and reagents for quantitating the amount of HA ina sample, such as, but not limited to, detection reagents, such asantibodies, buffers, substrates for enzymatic staining, chromogens orother materials, such as slides, containers, microtiter plates, andoptionally, instructions for performing the methods. Those of skill inthe art will recognize many other possible containers and plates andreagents that can be used for contacting the various materials. Kitsalso can contain control samples representing tissues with differentlevels of HA or reference samples stained for HA content for comparisonand classification of the test samples. The HABP diagnostic provided canbe provided in a lyophilized or other stable formulation of thediagnostic agent. In some examples, the kit includes a device, such asan automated cellular imaging system (ACIS) fluorometer, luminometer, orspectrophotometer for assay detection.

Also provided are combinations of an HABP reagent provided herein,including the improved HABP reagents provided, and a hyaluronandegrading enzyme. As described herein, HABPs can be employed ascompanion diagnostic agents for treatment with a hyaluronan degradingenzyme. Such combinations optionally can be packaged as kits for the foruse in selecting patients for treatment with an anti-hyaluronan agent(e.g. a hyaluronan degrading enzyme) and treating such patients with theanti-hyaluronan agent (e.g. a hyaluronan degrading enzyme), forpredicting the efficacy of treatment with an anti-hyaluronan agent (e.g.a hyaluronan degrading enzyme) in a patient and treating such patientswith the anti-hyaluronan agent (e.g. hyaluronan degrading enzyme), fordetermining the prognosis of a patient with an HA-associated diseasesand treating such patients with the anti-hyaluronan agent (e.g.hyaluronan degrading enzyme), or for monitoring the efficacy oftreatment of a patient with an anti-hyaluronan-degrading enzyme (e.g. ahyaluronan degrading enzyme) for the treatment of HA-associateddiseases, in particular cancer, and treating such patients with theanti-hyaluronan agent (e.g. hyaluronan degrading enzyme) based onefficacy of treatment. Combinations, which can be packaged as kits, caninclude, one or more additional agents for therapy, such as ananti-cancer agent or for the treatment or a side effect of therapy,including a corticosteroid for the treatment of musculoskeletal sideseffects associated with treatment with an anti-hyaluronan agent (e.g.hyaluronan degrading enzyme). The kits can include packing materials forthe packaging of the anti-hyaluronan agent (e.g. hyauronan degradingenzyme) or the one or more additional therapeutic agents. For example,the kits can contain containers including single chamber and dualchamber containers. The containers include, but are not limited to,tubes, bottles and syringes. The containers can further includematerials for administration, such as a needle for subcutaneousadministration. The anti-hyaluronan agent (e.g. a hyauronan degradingenzyme) or the one or more additional therapeutic agents can be providedtogether or separately. The kit can, optionally, include instructionsfor administration including dosages, dosing regimens and instructionsfor modes of administration.

Kits provided herein also can include reagents for detecting theexpression of one or more additional proteins or encoding RNAs in thesample, such as, for example, one or more additional cancer markers,such as, for example, but not limited to, carcinoembryonic antigen(CEA), Alpha-Fetoprotein (AFP), CA125, CA19-9, prostate specific antigen(PSA), human chorionic gonadotropin (HCG), HER2/neu antigen, CA27.29,CYFRA 21-2, LASA-P, CA15-3, TPA, S-100 and CA-125.

I. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 rHuPH20 Expressing Cell Lines

A. Generation of an Initial Soluble rHuPH20-Expressing Cell Line

Chinese Hamster Ovary (CHO) cells were transfected with the HZ24 plasmid(set forth in SEQ ID NO:52). The HZ24 plasmid vector for expression ofsoluble rHuPH20 contains a pCI vector backbone (Promega), DNA encodingamino acids 1-482 of human PH20 hyaluronidase (SEQ ID NO:49), aninternal ribosomal entry site (IRES) from the ECMV virus (Clontech), andthe mouse dihydrofolate reductase (DHFR) gene. The pCI vector backbonealso includes DNA encoding the Beta-lactamase resistance gene (AmpR), anf1 origin of replication, a Cytomegalovirus immediate-earlyenhancer/promoter region (CMV), a chimeric intron, and an SV40 latepolyadenylation signal (SV40). The DNA encoding the soluble rHuPH20construct contains an NheI site and a Kozak consensus sequence prior tothe DNA encoding the methionine at amino acid position 1 of the native35 amino acid signal sequence of human PH20, and a stop codon followingthe DNA encoding the tyrosine corresponding to amino acid position 482of the human PH20 hyaluronidase set forth in SEQ ID NO:1), followed by aBamHI restriction site. The construct pCI-PH20-IRES-DHFR-SV40pa (HZ24),therefore, results in a single mRNA species driven by the CMV promoterthat encodes amino acids 1-482 of human PH20 (set forth in SEQ ID NO:3and amino acids 1-186 of mouse dihydrofolate reductase (set forth in SEQID NO:53 separated by the internal ribosomal entry site (IRES).

Non-transfected CHO cells growing in GIBCO Modified CD-CHO media forDHFR(-) cells, supplemented with 4 mM Glutamine and 18 ml/L PluronicF68/L (Gibco), were seeded at 0.5×10⁶ cells/ml in a shaker flask inpreparation for transfection. Cells were grown at 37° C. in 5% CO₂ in ahumidified incubator, shaking at 120 rpm. Exponentially growingnon-transfected CHO cells were tested for viability prior totransfection.

Sixty million viable cells of the non-transfected CHO cell culture werepelleted and resuspended to a density of 2×10⁷ cells in 0.7 mL of 2×transfection buffer (2×HeBS: 40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mMKCl, 1.4 mM Na₂HPO₄, 12 mM dextrose). To each aliquot of resuspendedcells, 0.09 mL (250 μg) of the linear HZ24 plasmid (linearized byovernight digestion with Cla I (New England Biolabs) was added, and thecell/DNA solutions were transferred into 0.4 cm gap BTX (Gentronics)electroporation cuvettes at room temperature. A negative controlelectroporation was performed with no plasmid DNA mixed with the cells.The cell/plasmid mixes were electroporated with a capacitor discharge of330 V and 960 μF or at 350 V and 960 μF.

The cells were removed from the cuvettes after electroporation andtransferred into 5 mL of Modified CD-CHO media for DHFR(-) cells,supplemented with 4 mM Glutamine and 18 ml/L Pluronic F68/L (Gibco), andallowed to grow in a well of a 6-well tissue culture plate withoutselection for 2 days at 37° C. in 5% CO₂ in a humidified incubator.

Two days post-electroporation, 0.5 mL of tissue culture media wasremoved from each well and tested for the presence of hyaluronidaseactivity using the microturbidity assay described in Example 3. Cellsexpressing the highest levels of hyaluronidase activity were collectedfrom the tissue culture well, counted and diluted to 1×10⁴ to 2×10⁴viable cells per mL. A 0.1 mL aliquot of the cell suspension wastransferred to each well of five, 96 well round bottom tissue cultureplates. One hundred microliters of CD-CHO media (GIBCO) containing 4 mMGlutaMAX™-1 supplement (GIBCO™, Invitrogen Corporation) and withouthypoxanthine and thymidine supplements were added to the wellscontaining cells (final volume 0.2 mL).

Ten clones were identified from the 5 plates grown without methotrexate.Six of these HZ24 clones were expanded in culture and transferred intoshaker flasks as single cell suspensions. Clones 3D3, 3E5, 2G8, 2D9,1E11, and 4D10 were plated into 96-well round bottom tissue cultureplates using a two-dimensional infinite dilution strategy in which cellswere diluted 1:2 down the plate, and 1:3 across the plate, starting at5000 cells in the top left hand well. Diluted clones were grown in abackground of 500 non-transfected DG44 CHO cells per well, to providenecessary growth factors for the initial days in culture. Ten plateswere made per subclone, with 5 plates containing 50 nM methotrexate and5 plates without methotrexate.

Clone 3D3 produced 24 visual subclones (13 from the no methotrexatetreatment, and 11 from the 50 nM methotrexate treatment). Significanthyaluronidase activity was measured in the supernatants from 8 of the 24subclones (>50 Units/mL), and these 8 subclones were expanded into T-25tissue culture flasks. Clones isolated from the methotrexate treatmentprotocol were expanded in the presence of 50 nM methotrexate. Clone3D35M was further expanded in 500 nM methotrexate in shaker flasks andgave rise to clones producing in excess of 1,000 Units/ml hyaluronidaseactivity (clone 3D35M; or Gen1 3D35M). A master cell bank (MCB) of the3D35M cells was then prepared.

B. Generation of a Second Generation Cell Line Expressing SolublerHuPH20

The Gen1 3D35M cell line described in Example 1A was adapted to highermethotrexate levels to produce generation 2 (Gen2) clones. 3D35M cellswere seeded from established methotrexate-containing cultures into CDCHO medium containing 4 mM GlutaMAX-1™ and 1.0 μM methotrexate. Thecells were adapted to a higher methotrexate level by growing andpassaging them 9 times over a period of 46 days in a 37° C., 7% CO₂humidified incubator. The amplified population of cells was cloned outby limiting dilution in 96-well tissue culture plates containing mediumwith 2.0 μM methotrexate. After approximately 4 weeks, clones wereidentified and clone 3E10B was selected for expansion. 3E10B cells weregrown in CD CHO medium containing 4 mM GlutaMAX-1™ and 2.0 μMmethotrexate for 20 passages. A master cell bank (MCB) of the 3E10B cellline was created and frozen and used for subsequent studies.

Amplification of the cell line continued by culturing 3E10B cells in CDCHO medium containing 4 mM GlutaMAX-1™ and 4.0 μM methotrexate. Afterthe 12^(th) passage, cells were frozen in vials as a research cell bank(RCB). One vial of the RCB was thawed and cultured in medium containing8.0 μM methotrexate. After 5 days, the methotrexate concentration in themedium was increased to 16.0 μM, then 20.0 μM 18 days later. Cells fromthe 8^(th) passage in medium containing 20.0 μM methotrexate were clonedout by limiting dilution in 96-well tissue culture plates containing CDCHO medium containing 4 mM GlutaMAX-1™ and 20.0 μM methotrexate. Cloneswere identified 5-6 weeks later and clone 2B2 was selected for expansionin medium containing 20.0 μM methotrexate. After the 11th passage, 2B2cells were frozen in vials as a research cell bank (RCB).

The resultant 2B2 cells are dihydrofolate reductase deficient (dhfr-)DG44 CHO cells that express soluble recombinant human PH20 (rHuPH20).The soluble PH20 is present in 2B2 cells at a copy number ofapproximately 206 copies/cell. Southern blot analysis of Spe I-, Xba I-and BamH I/Hind III-digested genomic 2B2 cell DNA using arHuPH20-specific probe revealed the following restriction digestprofile: one major hybridizing band of ˜7.7 kb and four minorhybridizing bands (˜13.9, ˜6.6, ˜5.7 and ˜4.6 kb) with DNA digested withSpe I; one major hybridizing band of ˜5.0 kb and two minor hybridizingbands (˜13.9 and ˜6.5 kb) with DNA digested with Xba I; and one singlehybridizing band of ˜1.4 kb observed using 2B2 DNA digested with BamHI/Hind III. Sequence analysis of the mRNA transcript indicated that thederived cDNA (SEQ ID NO:56) was identical to the reference sequence (SEQID NO:49) except for one base pair difference at position 1131, whichwas observed to be a thymidine (T) instead of the expected cytosine (C).This is a silent mutation, with no effect on the amino acid sequence.

Example 2 Production and Purification of rHuPH20

A. Production of Gen2 Soluble rHuPH20 in 300 L Bioreactor Cell Culture

A vial of HZ24-2B2 cells (Example 1B) was thawed and expanded fromshaker flasks through 36 L spinner flasks in CD-CHO media (Invitrogen,Carlsbad, Calif.) supplemented with 20 μM methotrexate and GlutaMAX-1™(Invitrogen). Briefly, a vial of cells was thawed in a 37° C. waterbath, media was added and the cells were centrifuged. The cells werere-suspended in a 125 mL shake flask with 20 mL of fresh media andplaced in a 37° C., 7% CO₂ incubator. The cells were expanded up to 40mL in the 125 mL shake flask. When the cell density reached greater than1.5×10⁶ cells/mL, the culture was expanded into a 125 mL spinner flaskin a 100 mL culture volume. The flask was incubated at 37° C., 7% CO₂.When the cell density reached greater than 1.5×10⁶ cells/mL, the culturewas expanded into a 250 mL spinner flask in 200 mL culture volume, andthe flask was incubated at 37° C., 7% CO₂. When the cell density reachedgreater than 1.5×10⁶ cells/mL, the culture was expanded into a 1 Lspinner flask in 800 mL culture volume and incubated at 37° C., 7% CO₂.When the cell density reached greater than 1.5×10⁶ cells/mL the culturewas expanded into a 6 L spinner flask in 5000 mL culture volume andincubated at 37° C., 7% CO₂. When the cell density reached greater than1.5×10⁶ cells/mL the culture was expanded into a 36 L spinner flask in32 L culture volume and incubated at 37° C., 7% CO₂.

A 400 L reactor was sterilized and 230 mL of CD-CHO media was added.Before use, the reactor was checked for contamination. Approximately 30L cells were transferred from the 36 L spinner flasks to the 400 Lbioreactor (Braun) at an inoculation density of 4.0×10⁵ viable cells perml and a total volume of 260 L. Parameters were temperature set point,37° C.; Impeller Speed 40-55 RPM; Vessel Pressure: 3 psi; Air Sparge0.5-1.5 L/Min.; Air Overlay: 3 L/min. The reactor was sampled daily forcell counts, pH verification, media analysis, protein production andretention. Also, during the run nutrient feeds were added. At 120 hrs(day 5), 10.4 L of Feed #1 Medium (4×CD-CHO+33 g/L Glucose+160 mL/LGlutamax-1™+83 mL/L Yeastolate+33 mg/L rHuInsulin) was added. At 168hours (day 7), 10.8 L of Feed #2 (2×CD-CHO+33 g/L Glucose+80 mL/LGlutamax-1™+167 mL/L Yeastolate+0.92 g/L Sodium Butyrate) was added, andculture temperature was changed to 36.5° C. At 216 hours (day 9), 10.8 Lof Feed #3 (1×CD-CHO+50 g/L Glucose+50 mL/L Glutamax-1™+250 mL/LYeastolate+1.80 g/L Sodium Butyrate) was added, and culture temperaturewas changed to 36° C. At 264 hours (day 11), 10.8 L of Feed #4(1×CD-CHO+33 g/L Glucose+33 mL/L Glutamax-1™+250 mL/L Yeastolate+0.92g/L Sodium Butyrate) was added, and culture temperature was changed to35.5° C. The addition of the feed media was observed to dramaticallyenhance the production of soluble rHuPH20 in the final stages ofproduction. The reactor was harvested at 14 or 15 days or when theviability of the cells dropped below 40%. The process resulted in afinal productivity of 17,000 Units per ml with a maximal cell density of12 million cells/mL. At harvest, the culture was sampled for mycoplasma,bioburden, endotoxin and viral in vitro and in vivo, TransmissionElectron Microscopy (TEM) and enzyme activity.

The culture was pumped by a peristaltic pump through four Millistakfiltration system modules (Millipore) in parallel, each containing alayer of diatomaceous earth graded to 4-8 μm and a layer of diatomaceousearth graded to 1.4-1.1 μm, followed by a cellulose membrane, thenthrough a second single Millistak filtration system (Millipore)containing a layer of diatomaceous earth graded to 0.4-0.11 μm and alayer of diatomaceous earth graded to <0.1 μm, followed by a cellulosemembrane, and then through a 0.22 μm final filter into a sterile singleuse flexible bag with a 350 L capacity. The harvested cell culture fluidwas supplemented with 10 mM EDTA and 10 mM Tris to a pH of 7.5. Theculture was concentrated 10× with a tangential flow filtration (TFF)apparatus using four Sartoslice TFF 30 kDa molecular weight cut-off(MWCO) polyether sulfone (PES) filters (Sartorius), followed by a 10×buffer exchange with 10 mM Tris, 20 mM Na₂SO₄, pH 7.5 into a 0.22 μmfinal filter into a 50 L sterile storage bag.

The concentrated, diafiltered harvest was inactivated for virus. Priorto viral inactivation, a solution of 10% Triton X-100, 3%tri(n-butyl)phosphate (TNBP) was prepared. The concentrated, diafilteredharvest was exposed to 1% Triton X-100, 0.3% TNBP for 1 hour in a 36 Lglass reaction vessel immediately prior to purification on the Q column.

B. Purification of Gen2 Soluble rHuPH20

A Q Sepharose (Pharmacia) ion exchange column (9 L resin, H=29 cm, D=20cm) was prepared. Wash samples were collected for a determination of pH,conductivity and endotoxin (LAL) assay. The column was equilibrated with5 column volumes of 10 mM Tris, 20 mM Na2SO4, pH 7.5. Following viralinactivation, the concentrated, diafiltered harvest (Example 2A) wasloaded onto the Q column at a flow rate of 100 cm/hr. The column waswashed with 5 column volumes of 10 mM Tris, 20 mM Na2SO4, pH 7.5 and 10mM Hepes, 50 mM NaCl, pH 7.0. The protein was eluted with 10 mM Hepes,400 mM NaCl, pH 7.0 into a 0.22 μm final filter into sterile bag. Theeluate sample was tested for bioburden, protein concentration andhyaluronidase activity. A₂₈₀ absorbance readings were taken at thebeginning and end of the exchange.

Phenyl-Sepharose (Pharmacia) hydrophobic interaction chromatography wasnext performed. A Phenyl-Sepharose (PS) column (19-21 L resin, H=29 cm,D=30 cm) was prepared. The wash was collected and sampled for pH,conductivity and endotoxin (LAL assay). The column was equilibrated with5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate,0.1 mM CaCl2, pH 7.0. The protein eluate from the Q sepharose column wassupplemented with 2M ammonium sulfate, 1 M potassium phosphate and 1 MCaCl₂ stock solutions to yield final concentrations of 5 mM, 0.5 M and0.1 mM, respectively. The protein was loaded onto the PS column at aflow rate of 100 cm/hr and the column flow thru collected. The columnwas washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate and 0.1mM CaCl2 pH 7.0 at 100 cm/hr and the wash was added to the collectedflow thru. Combined with the column wash, the flow through was passedthrough a 0.22 μm final filter into a sterile bag. The flow through wassampled for bioburden, protein concentration and enzyme activity.

An aminophenyl boronate column (ProMedics) was prepared. The wash wascollected and sampled for pH, conductivity and endotoxin (LAL assay).The column was equilibrated with 5 column volumes of 5 mM potassiumphosphate, 0.5 M ammonium sulfate. The PS flow through containingpurified protein was loaded onto the aminophenyl boronate column at aflow rate of 100 cm/hr. The column was washed with 5 mM potassiumphosphate, 0.5 M ammonium sulfate, pH 7.0. The column was washed with 20mM bicine, 0.5 M ammonium sulfate, pH 9.0. The column was washed with 20mM bicine, 100 mM sodium chloride, pH 9.0. The protein was eluted with50 mM Hepes, 100 mM NaCl, pH 6.9 and passed through a sterile filterinto a sterile bag. The eluted sample was tested for bioburden, proteinconcentration and enzyme activity.

The hydroxyapatite (HAP) column (Biorad) was prepared. The wash wascollected and tested for pH, conductivity and endotoxin (LAL assay). Thecolumn was equilibrated with 5 mM potassium phosphate, 100 mM NaCl, 0.1mM CaCl₂, pH 7.0. The aminophenyl boronate purified protein wassupplemented to final concentrations of 5 mM potassium phosphate and 0.1mM CaCl₂ and loaded onto the HAP column at a flow rate of 100 cm/hr. Thecolumn was washed with 5 mM potassium phosphate, pH 7, 100 mM NaCl, 0.1mM CaCl₂. The column was next washed with 10 mM potassium phosphate, pH7, 100 mM NaCl, 0.1 mM CaCl₂. The protein was eluted with 70 mMpotassium phosphate, pH 7.0 and passed through a 0.22 μm sterile filterinto a sterile bag. The eluted sample was tested for bioburden, proteinconcentration and enzyme activity.

The HAP purified protein was then passed through a viral removal filter.The sterilized Viosart filter (Sartorius) was first prepared by washingwith 2 L of 70 mM potassium phosphate, pH 7.0. Before use, the filteredbuffer was sampled for pH and conductivity. The HAP purified protein waspumped via a peristaltic pump through the 20 nM viral removal filter.The filtered protein in 70 mM potassium phosphate, pH 7.0 was passedthrough a 0.22 μm final filter into a sterile bag. The viral filteredsample was tested for protein concentration, enzyme activity,oligosaccharide, monosaccharide and sialic acid profiling. The samplealso was tested for process related impurities.

Example 3 Determination of Hyaluronidase Activity of Soluble rHuPH20

Hyaluronidase activity of soluble rHuPH20 in samples such as cellcultures, plasma, purification fractions and purified solutions wasdetermined using either a turbidimetric assay, which is based on theformation of an insoluble precipitate when hyaluronic acid binds withserum albumin, or a biotinylated-hyaluronic acid substrate assay, whichmeasures the amount of enzymatically active rHuPH20 or PEGPH20 by thedigestion of biotinylated hyaluronic acid (b-HA) substratenon-covalently bound to plastic multi-well microtiter plates.

A. Microturbidity Assay

Hyaluronidase activity of soluble rHuPH20 is measured by incubatingsoluble rHuPH20 with sodium hyaluronate (hyaluronic acid) for a setperiod of time (10 minutes) and then precipitating the undigested sodiumhyaluronate with the addition of acidified serum albumin. The turbidityof the resulting sample is measured at 640 nm after a 30 minutedevelopment period. The decrease in turbidity resulting from enzymeactivity on the sodium hyaluronate substrate is a measure of the solublerHuPH20 hyaluronidase activity. The method is performed using acalibration curve generated with dilutions of a soluble rHuPH20 assayworking reference standard, and sample activity measurements are maderelative to this calibration curve.

Dilutions of the sample were prepared in Enzyme Diluent Solutions. TheEnzyme Diluent Solution was prepared by dissolving 33.0±0.05 mg ofhydrolyzed gelatin in 25.0 mL of the 50 mM PIPES Reaction Buffer (140 mMNaCl, 50 mM PIPES, pH 5.5) and 25.0 mL of sterile water for injection(SWFI), and diluting 0.2 mL of 25% Buminate solution into the mixtureand vortexing for 30 seconds. This was performed within 2 hours of useand stored on ice until needed. The samples were diluted to an estimated1-2 U/mL. Generally, the maximum dilution per step did not exceed 1:100and the initial sample size for the first dilution was not less than 20μL. The minimum sample volumes needed to perform the assay were asfollows: In-process Samples, FPLC Fractions: 80 μL; Tissue CultureSupernatants: 1 mL; Concentrated Material: 80 μL; Purified or Final StepMaterial: 80 μL. The dilutions were made in triplicate in a Low ProteinBinding 96-well plate, and 30 μL of each dilution was transferred toOptilux black/clear bottom plates (BD BioSciences).

Dilutions of known soluble rHuPH20 with a concentration of 2.5 U/mL wereprepared in Enzyme Diluent Solution to generate a standard curve andadded to the Optilux plate in triplicate. The dilutions included 0 U/mL,0.25 U/mL, 0.5 U/mL, 1.0 U/mL, 1.5 U/mL, 2.0 U/mL, and 2.5 U/mL.“Reagent blank” wells that contained 60 μL of Enzyme Diluent Solutionwere included in the plate as a negative control. The plate was thencovered and warmed on a heat block for 5 minutes at 37° C. The cover wasremoved and the plate was shaken for 10 seconds. After shaking, theplate was returned to the heat block and the MULTIDROP 384 LiquidHandling Device was primed with the warm 0.25 mg/mL sodium hyaluronatesolution (prepared by dissolving 100 mg of sodium hyaluronate (LifeCoreBiomedical) in 20.0 mL of SWFI. This was mixed by gently rotating and/orrocking at 2-8° C. for 2-4 hours, or until completely dissolved). Thereaction plate was transferred to the MULTIDROP 384 and the reaction wasinitiated by pressing the start key to dispense 30 μL sodium hyaluronateinto each well. The plate was then removed from the MULTIDROP 384 andshaken for 10 seconds before being transferred to a heat block with theplate cover replaced. The plate was incubated at 37° C. for 10 minutes.

The MULTIDROP 384 was prepared to stop the reaction by priming themachine with Serum Working Solution and changing the volume setting to240 μL. (25 mL of Serum Stock Solution [1 volume of Horse Serum (Sigma)was diluted with 9 volumes of 500 mM Acetate Buffer Solution and the pHwas adjusted to 3.1 with hydrochloric acid] in 75 mL of 500 mM AcetateBuffer Solution). The plate was removed from the heat block and placedonto the MULTIDROP 384, and 240 μL of serum Working Solutions wasdispensed into the wells. The plate was removed and shaken on a platereader for 10 seconds. After a further 15 minutes, the turbidity of thesamples was measured at 640 nm and the hyaluronidase activity (in U/mL)of each sample was determined by fitting to the standard curve.

Specific activity (Units/mg) was calculated by dividing thehyaluronidase activity (U/ml) by the protein concentration (mg/mL).

B. Biotinylated Hyaluronan Assay

The biotinylated-hyaluronic acid assay measures the amount ofenzymatically active rHuPH20 or PEGPH20 in biological samples by thedigestion of a large molecular weight (˜1.2 megadaltons) biotinylatedhyaluronic acid (b-HA) substrate non-covalently bound to plasticmulti-well microtiter plates. The rHuPH20 or PEGPH20 in standards andsamples are allowed to incubate in a plate coated with b-HA at 37° C.After a series of washes, remaining uncleaved/bound b-HA is treated withStreptavidin Horseradish Peroxidase conjugate (SA-HRP). Reaction betweenimmobilized SA-HRP and the chromogenic substrate,3,3′,5,5′-tetramethylbenzidine (TMB), produces a blue colored solution.After stopping the reaction with acid, formation of the soluble yellowreaction product is determined by reading the absorbance at 450 nm usinga microtiter plate spectrophotometer. The decrease in absorbance at 450nm resulting from enzyme activity on the biotinylated hyaluronic acid(b-HA) substrate is a measure of the soluble rHuPH20 hyaluronidaseactivity. The method is performed using a calibration curve generatedwith dilutions of a soluble rHuPH20 or PEGPH20 reference standard, andsample activity measurements are made relative to this calibrationcurve.

Dilutions of the sample and calibrator were prepared in Assay Diluent.The Assay Diluent was prepared by adding 1% v/v pooled plasma (from theappropriate species) to 0.1% (w/v) BSA in HEPES, pH 7.4. This wasprepared daily and stored at 2-8° C. Depending upon the species type aswell as the anticipated hyaluronidase level, single or multipledilutions were prepared to ensure at least one sample dilution wouldfall within the range of the calibration curve. To guide the selectionof test sample dilution(s), information known about the dose ofhyaluronidase administered, the route of administration, approximateplasma volume of the species and the time point were used to estimatethe hyaluronidase activity levels. Each sample dilution was mixed as itwas prepared by brief pulse-vortexing and pipet tips were changed inbetween each dilution. In general, the dilutions began with an initial50 or 100-fold dilution followed by additional serial dilutions. Aseven-point calibration curve of rHuPH20 or PEGPH20 (depending upon thetreatment administered) was prepared ranging in concentration from 0.004to 3.0 U/mL for rHuPH20 and from 0.037 to 27 U/mL for PEGPH20.One-hundred microliters (100 μL) of each test sample dilution andcalibration curve point was applied to triplicate wells of a 96-wellmicrotiter plate (Immulon 4HBX, Thermo) that had been previously coatedwith 100 μL per well of b-HA at 0.1 mg/mL and blocked with 250 μL of1.0% (w/v) Bovine Serum Albumin in PBS. Plate(s) were covered with anadhesive plate seal and incubated at 37° C. for approximately 90minutes. At the end of the incubation period, the adhesive seal wasremoved from the plate, samples were aspirated and the plate washed five(5) times with 300 μL per well Wash Buffer (10 mM Phosphate Buffer, 2.7mM Potassium Chloride, 137 mM Sodium Chloride, pH 7.4, with 0.05% (v/v)Tween 20, PBST) using an automated plate washer (BioTek ELx405 SelectCW, Program ‘4HBX1’). One hundred microliters of Streptavidin-HRPConjugate Working Solution [Streptavidin-HRP conjugate (1:5,000 v/v) in20 mM Tris-HCl, 137 mM Sodium Chloride, 0.025% (v/v) Tween 20, 0.1%(w/v) Bovine Serum Albumin] was added per well. The plate was sealed andallowed to incubate at ambient temperature for approximately 60 minuteswithout shaking and protected from light. At the end of the incubationperiod, the adhesive seal was removed from the plate, samples wereaspirated and the plate washed five (5) times with 300 μL per well WashBuffer as described above. TMB solution (at ambient temperature) wasadded to each well and allowed to incubate protected from light forapproximately five (5) minutes at room temperature. TMB Stop Solution(KPL, Catalog #50-85-06) was then added as 100 μL per well. Theabsorbance of each well at 450 nm was determined using a microtiterplate spectrophotometer. The response of the Calibration Curve on eachplate was modeled using a 4-parameter logistic curve fit. Thehyaluronidase activity of each unknown was calculated by interpolationfrom the calibration curve, corrected for sample dilution factor, andreported in U/mL.

Example 4 Preparation of PEGylated rHuPH20

In this example, rHuPH20 was PEGylated by reaction of the enzyme withlinear N-hydroxysuccinimidyl ester of methoxy poly(ethylene glycol)butanoic acid (mPEG-SBA-30K).

A. Preparation of mPEG-SBA-30K

In order to generate PEGPH20, rHuPH20 (which is approximately 60 KDa insize) was covalently conjugated to a linear N-hydroxysuccinimidyl esterof methoxy poly(ethylene glycol) butanoic acid (mPEG-SBA-30K), having anapproximate molecular weight of 30 kDa. The structure of mPEG-SBA isshown below, where n≈681.

Methods used to prepare the mPEG-SBA-30K that was used to PEGylaterHuPH20 are described, for example, in U.S. Pat. No. 5,672,662. Briefly,the mPEG-SBA-30K is made according to the following procedure:

A solution of ethyl malonate (2 equivalents) dissolved in dioxane isadded drop by drop to sodium hydride (2 equivalents) and toluene under anitrogen atmosphere. mPEG methane sulfonate (1 equivalent, MW 30 kDa,Shearwater) is dissolved in toluene and added to the above mixture. Theresulting mixture is refluxed for approximately 18 hours. The reactionmixture is concentrated to half its original volume, extracted with 10%aqueous NaCl solution, extracted with 1% aqueous hydrochloric acid, andthe aqueous extracts are combined. The collected aqueous layers areextracted with dichloromethane (3×) and the organic layer is dried withmagnesium sulfate, filtered and evaporated to dryness. The resultingresidue is dissolved in 1N sodium hydroxide containing sodium chlorideand the mixture is stirred for 1 hour. The pH of the mixture is adjustedto approximately 3 by addition of 6N hydrochloric acid. The mixture isextracted with dichloromethane (2×).

The organic layer is dried over magnesium sulfate, filtered,concentrated, and poured into cold diethyl ether. The precipitate iscollected by filtration and dried under vacuum. The resulting compoundis dissolved in dioxane and refluxed for 8 hours and then concentratedto dryness. The resulting residue is dissolved in water and extractedwith dichloromethane (2×), dried over magnesium sulfate, and thesolution is concentrated by rotary evaporation and then poured into colddiethyl ether. The precipitate is collected by filtration and driedunder vacuum. The resulting compound (1 equivalent) is dissolved indichloromethane and N-hydroxysuccinimide (2.1 equivalents) is added. Thesolution is cooled to 0° C. and a solution of dicyclohexylcarbodiimide(2.1 equivalents) in dichloromethane is added dropwise. The solution isstirred at room temperature for approximately 18 hours. The reactionmixture is filtered, concentrated and precipitated in diethyl ether. Theprecipitate is collected by filtration and dried under vacuum to affordthe powder mPEG-SBA-30K which is then frozen at ≦−15° C.

B. Conjugation of mPEG-SBA-30K to rHuPH20

To make the PEGPH20, mPEG-SBA-30K was coupled to the amino group(s) ofrHuPH20 by covalent conjugation, providing stable amide bonds betweenrHuPH20 and mPEG, as shown below, where n 681.

Prior to conjugation, the rHuPH20 purified bulk protein made in Example2B was concentrated to 10 mg/mL, using a 10 kDa polyethersulfone (PES)tangential flow filtration (TFF) cassettes (Sartorius) with a 0.2 m²filtration area, and buffer exchanged against 70 mM Potassium Phosphateat pH 7.2. The concentrated protein was then stored at 2-8° C. untiluse.

To conjugate the rHuPH20, the mPEG-SBA-30K (Nektar) was thawed at roomtemperature in the dark for not longer than 2 hours. Depending on thebatch size, a sterile 3″ stir bar was placed into a 1 or 3 literErlenmeyer flask and buffer exchanged rHuPH20 protein was added. Fivegrams of dry mPEG-SBA-30K powder per gram of rHuPH20 (10:1 molar ratioof mPEG-SBA-30K:rHuPH20) was added to the flask under a vaccuum hood andthe mixture was mixed for 10 minutes or until the mPEG-SBA-30K wascomplete dissolved. The stir rate was set such that vortexing occurredwithout foaming.

The solution was then filtered under a class 100 hood by pumping thesolution, via peristaltic pump, through a 0.22 μm polystyrene, celluloseacetate filter capsule (Corning 50 mL Tubetop filter) into a new 1 or 3liter Erlenmeyer flask containing a sterile 3″ stir bar. The volume ofthe PEGPH20 reaction mixture was determined by mass (1 g/mL density) andthe 0.22 μm filter used for filtration was examined in a post-useintegrity test.

The mixture was then placed on a stir plate at 2-8° C. and mixed for20±1 hours in the dark. The stir rate was again set such that vortexingoccurred without foaming. The entire Erlenmeyer container was wrapped infoil to protect the solution from light. After mixing, the reaction wasquenched by adding 1M glycine to a final concentration of 25 mM. Sampleswere removed from the container to test pH and conductivity. The pH andconductivity were then adjusted by adding to a solution of 5 mM TrisBase (5.65 L/L) and 5 mM Tris, 10 mM NaCl, pH 8.0 (13.35 L/L) to proceedwith Q Sepharose purification.

A QFF Sepharose (GE Healthcare) ion exchange column (Height=21.5-24.0cm, Diameter=20 cm) was prepared by equilibration with 5 column volumes(36 L) of 5 mM Tris, 10 mM NaCl, pH 8.0. The conjugated product wasloaded onto the QFF column at a flow rate of 95 cm/hr. The column wasthen washed with 11 L of equilibration buffer (5 mM Tris, 10 mM NaCl, pH8.0) at a flow rate of 95 cm/hr followed by a wash with 25 L ofequilibration buffer at a flow rate of 268 cm/hr. The protein productwas then eluted with 5 mM Tris, 130 mM NaCl, pH 8.0 at a flow rate of268 cm/hr. The resulting purified PEGPH20 was concentrated to 3.5 mg/mL,using a 30 kDa polyethersulfone (PES) tangential flow filtration (TFF)cassettes (Sartorius) with a 0.2 m² filtration area, and bufferexchanged against 10 mM Histidine, 130 mM NaCl at pH 6.5. The resultingmaterial was tested for enzyme activity as described in Example 3. ThePEGylated rHuPH20 material at a concentration of 3.5 mg/mL (final enzymeactivity 140,000 U/mL) was filled, in 3 mL volumes, into 5 mL glassvials with a siliconized bromobutyl rubber stopper and aluminum flip-offseal, and frozen (frozen overnight in a −20° C. freezer, then put in a−80° C. freezer for longer storage). The PEGylated rHuHP20 containedapproximately 4.5 moles of PEG per mole of rHuPH20.

B. Analysis of PEGylated rHuPH20

The PEGylated rHuPH20 (PEGPH20) material was assayed by gelelectrophoresis. Three batches of PEGPH20, made as described in Example4A above, revealed an identical pattern of multiple bands, representingunreacted PEG and multiple species of PEGPH20 conjugates, which migratedat different distances. Based on comparison with migration of amolecular weight marker, the bands representing the species ranged fromapproximately 90 KDa to 300 KDa, with three dark bands migrating abovethe 240 KDa marker. These data indicated that the PEGPH20, generated bycovalent conjugation of mPEG-SBA-30K, contained a heterogeneous mixtureof PEGPH20 species, likely including mono-, di- and tri-PEGylatedproteins. The lack of a visible band at 60 KDa suggested that all theprotein had reacted with the PEG, and that no detectable native rHuPH20was present in the mixture.

Example 5 Competency of Tumor Cells to Form Pericellular Matrix andRelationship to Tumor Cell Hyaluronan (HA) Content, Levels of HyaluronanSynthase (HAS), and Hyaluronidase (Hyal) Expression A. Comparison ofTumor Cell HA Content, Expression of HAS 1, 2, 3 and Hyal 1 and 2, andPericellular Matrix Formation

In this Example, the amount of endogenous HA synthesis enzymes,hyaluronan synthase (HAS) 1, 2, and 3, hyaluronidase (Hyal) 1 and (Hyal)2 and the amount of hyaluronan (HA) accumulation in tumor cells wascompared to show that each correlated to pericellular matrix formationby the tumor cells.

1. Cell Lines Used in the Study

Ten cell lines from tumors of various tissue origin (e.g., prostate,breast, ovarian, pancreatic, and lung) and species origin (e.g., human,mouse and rat) were examined in the study. The following cell lines wereobtained from the American Type Culture Collection (ATCC): 4T1 mousebreast tumor (ATCC CRL-2539), PC-3 human prostate adenocarcinoma (ATCCCRL-1435), BxPC-3 human pancreatic adenocarcinoma (ATCC CRL-1687), MDAMB 231 human breast adenocarcinoma (ATCC HTB-26), Mat-Lylu rat malignantprostate carcinoma (ATCC JHU-92), AsPc-1 human pancreatic adenocarcinoma(ATCC CRL-1682), DU-145 human prostate carcinoma (ATCC HTB-81), and MIAPaCa 2 human pancreatic carcinoma (ATCC CRL-1420). The ATCC cell lineswere grown in recommended culture medium containing 10% FBS at 37° C. ina humidified incubator supplied with 5% CO₂/95% air. MDA-MB-231-Luc(Cat. No. D3H2LN) cells, which express the North American FireflyLuciferase gene, were purchased from Caliper Life Sciences Inc. andgrown in RPMI containing 10% FBS.

The DU-145/HAS2 and MDA-MB-231-Luc/HAS2 cell lines were generated bytransduction of the DU-145 and MDA-MB-231-Luc cell lines with aretrovirus encoding hyaluronan synthase 2 (HAS2) (SEQ ID NO:195). Togenerate the HAS2 retrovirus, N-terminal His6-tagged hHAS2 cDNA (SEQ IDNO:196) was inserted into the AvrII and NotI sites of the vector pLXRN(SEQ ID NO:197; Clontech, Cat. No. 631512), which includes the neomycinresistance gene, to create pLXRN-hHAS2 (SEQ ID NO: 201). The pLXRN-hHAS2His plasmid was then co-transfected with pVSV-G envelope vector (SEQ IDNO:198 Clontech, part of Cat. No. 631530) into GP-293 cells usingLipofectamine 2000 reagent (Life Technologies). A DU-145 Mock cell linealso was generated by co-transfection of the empty pLXRN plasmid andpVSV-G envelope vector.

The virus titer was determined by quantitative PCR method (Retro-X™qRT-PCR Titration Kit; Clontech, Catalog No. 631453) using the followingprimers (Clontech Catalog No. #K1060-E):

pLXSN 5′primer (1398-1420): (SEQ ID NO: 199)5′-CCCTTGAACCTCCTCGTTCGACC-3′; pLXSN 3′ primer (1537-1515):(SEQ ID NO: 200) 5′-GAGCCTGGGGACTTTCCACACCC-3′.

To establish HAS2 expression cell lines, 70% confluent cancer cells,DU-145 or MDA MB 231 Luc, were incubated with a 60:1 to 6:1 ratio ofretrovirus in DMEM (Mediatech) containing 10% FBS for 72 hours. Thecultures were maintained in selective medium containing 200 μg/mL ofG418. Stable HAS2-expressing cancer cells were generated after 2 weeksof G418 conditional medium selection.

2. Quantification of Hyaluronic Acid

A hyaluronan binding protein (HABP)-based assay was employed todetermine the amount of hyaluronan produced by the tumor cells.HABP-based assays are preferable to chemical methods for measuring HA asa tumor microenvironment (TME) biomarker because the HABP preferentiallydetects HA composed of at least 15 (n-acetyl glucose-glucuronic acid)disaccharides, which is competent to bind hyaladherins (HA bindingproteins) (see, e.g., Haserodt S, et al. (2011) Glycobiology 21:175-183).

Tumor cells were seeded at 1×10⁶ cells in 75 cm² flasks and incubatedfor 24 hours. Tissue culture supernatants were harvested forquantitation of HA using an enzyme-linked HABP sandwich assay (R&DSystems, Catalog No. DY3614), which uses recombinant human aggrecan asan HA capture and detection reagent (recombinant human aggrecanG1-IGD-G2 domains, Va120-Gly676 of Accession No. NP_(—)037359 (SEQ IDNO: 202) with a C-terminal 10-HIS tag, R&D Systems, Catalog No.1220-PG). The assay for HA detection was performed according to themanufacturer's instructions. Briefly, assay plates were coated withrecombinant human aggrecan, and samples (i.e. tissue culturesupernatants) containing HA were added to the plate (three independentreplicates of each cell line were tested). The plates were washed andthe bound HA was detected using biotinylated recombinant human aggrecan.After removing the unbound probe, streptavidin conjugated to horseradishperoxidase (HRP) was added as a secondary detection reagent. Afterwashing the plate, the bound HRP was detected by incubation with the 1:1H₂O₂/Tetramethylbenzidine substrate solution (R&D Systems) andquantitated by optical density detection at 450 nm using a SpectraMax M3Multi-Mode Microplate Reader (Molecular Devices, CA). Concentration ofHA in the culture media for each tumor cell type was expressed as meanHA concentration (ng/mL) in culture media (Table 5).

3. Quantification of HAS1, HAS2, HAS3, HYAL1 and HYAL2 mRNA Expression

RNA was extracted from cell pellets using an RNeasy® Mini Kit (QiagenGmbH) according to the manufacturer's instructions. The extracted RNAwas then quantified using a NanoDrop spectrophotometer (NanoDropTechnologies, Wilmington, Del.). Quantitative real-time PCR (qRT-PCR)using gene-specific primers was used to quantitate the relative mRNAlevels of each hyaluronan synthase and hyaluronidase. qRT-PCR primerswere purchased from Bio Applied Technologies Joint, Inc, (San Diego,Calif.). The DNA sequences for the primers used in the individual PCRreactions were as follows:

TABLE 4 Primer sequences used for qRT-PCR analysis of HAS and HYALgene expression Gene Forward primer Reverse primer HAS15′-TACAACCAGAAGTTCCTGGG-3′ 5′-CTGGAGGTGTACTTGGTAGC-3′ (SEQ ID NO: 395)(SEQ ID NO: 396) HAS2 5′-GTATCAGTTTGGTTTACAATC-3′5′-GCACCATGTCATATTGTTGTC-3′ (SEQ ID NO: 397) (SEQ ID NO: 398) HAS35′-CTTAAGGGTTGCTTGCTTGC-3′ 5′-GTTCGTGGGAGATGAAGGAA-3′ (SEQ ID NO: 399)(SEQ ID NO: 400) HYAL1 5′-GTGCTGCCCTATGTCCAGAT-3′5′-ATTTTCCCAGCTCACCCAGA-3′ (SEQ ID NO: 401) (SEQ ID NO: 402) HYAL25′-TCTACCATTGGCGAGAGTG-3′ 5′-GCAGCCGTGTCAGGTAAT-3′ (SEQ ID NO: 403)(SEQ ID NO: 404) GAPDH 5′-TGCACCACCAACTGCTTAGC-3′5′-GGCATGGACTGTGGTCATGAG-3′ (SEQ ID NO: 405) (SEQ ID NO: 406)

For the PCR reactions, samples were mixed with iQ SYBR Green master mix(Bio-Rad) and the designated primer pairs for each gene. The PCRreactions were performed on a Bio-Rad Chromo 4 qPCR device. First strandsynthesis was performed under the following conditions: 42° C. for 2minutes for the DNA elimination reaction, 42° C. for 15 minutes forreverse-transcription, and 3 minutes at 95° C. for inactivation ofreverse-transcriptase. For amplification, 3 minutes initial denaturationat 95° C., 45 cycles of 15 seconds denaturation and 1 minute annealingextension at 58° C. were used. The gene expression CT value from eachsample was calculated by normalizing with the internal housekeeping geneGAPDH and relative values were plotted. Table 5 lists the CT values foreach tumor cell type for each gene assayed.

4. Assay for Pericellular Matrix Formation

Monolayer cultures of the ten cell lines were grown and tested foraggrecan-facilitated pericellular matrix formation. To visualizeaggrecan-mediated HA pericellular matrices in vitro, particle exclusionassays were used as previously described in Thompson C B, et al. (2010)Mol Cancer Ther 9: 3052-3064, with some modifications. Briefly, cellswere seeded at 1×10⁵ cells per well in a six-well plate for 24 hours,and then treated with culture cell media alone or media containing 1000U/mL rHuPH20 at 37° C. for 1 hour. Pre-treatment with rHuPH20 inhibitsformation of the pericellular matrix; thus, it was employed as anegative control for pericellular matrix formation for each cell type.The cells were then incubated with 0.5 mg/mL of bovine aggrecan(Sigma-Aldrich) at 37° C. for 1 hour. Subsequently, media were removedand replaced with 10⁸/mL suspension of 2% glutaraldehyde-fixed mouse redblood cells (RBCs), isolated from Balb/c mouse (Taconic, Hudson, N.Y.),in PBS, pH 7.4. The particles were allowed to settle for 15 minutes. Thecultures were then imaged with a phase-contrast microscope coupled witha camera scanner and imaging program (Diagnostic Instruments). Particleexclusion area and cell area were measured using the SPOT Advanceprogram (Diagnostic Instruments, Inc., Sterling Heights, Mich.).Pericellular matrix area was calculated as matrix area minus cell area,and expressed as μm² (Table 5).

5. Results: Comparison of Tumor Cell HA Content, and HAS and HYALExpression to Pericellular Matrix Formation

The concentration of HA in conditioned media as determined by theHABP-based detection assay was found to correlate with the area ofaggrecan-mediated pericellular matrix formed by the tumor cells inmonolayer culture (Table 5, P<0.0029). Further, cell lines that wereengineered to express hyaluronan synthase 2 (HAS2), DU-145/HAS2 andMDA-MB-231/HAS2, displayed increased HA production and enhancedpericellular matrix formation in vitro compared to the respectiveparental cell lines. In contrast, no correlation was found betweenpericellular matrix formation and relative levels of HAS 1, 2, or 3 orHyal 1 or 2 mRNA expression. These findings indicate that the directmeasurement of tumor cell-associated HA specifically provides apredictor for pericellular matrix formation.

TABLE 5 Quantitation of HA production, pericellular matrix formation,HAS and Hyal expression in tumor cell lines Hyaluronidase HAS isoformmRNA³ isoform mRNA⁴ Tumor Cell Line PM¹ HA in CM² HAS1 HAS2 HAS3 Hyal1Hyal2 4T1 1552.00 473.83 NE NE NE NE NE MDA-MB-231/HAS2 1088.55 372.202.48 19.90 0.09 0.14 0.53 PC3 1072.20 294.45 1.41 0.34 6.32 0.14 1.19DU-145/HAS2 981.00 7417.00 1.08 7.81 0.65 0.34 1.04 BxPC3 967.20 467.121.00 1.00 1.00 1.00 1.00 MDA-MB-231 WT 770.45 256.90 3.39 0.54 0.05 0.130.64 MatLylu 760.55 265.91 NE NE NE NE NE AsPC-1 524.20 66.47 1.87 1.651.28 0.81 1.91 DU-145 WT 252.10 41.79 1.01 0.03 1.51 0.17 0.70 MIAPaCa-2 129.40 0.00 0.46 0.00 0.04 0.28 0.72 Correlation Coefficient —0.0029 0.23 0.34 0.71 0.66 0.36 (Spearman P value) NE: not evaluated¹Pericellular matrix area (μm³) assessed via particle exclusion assay.²Mean HA concentration (ng/mL) in culture media (n = 3, independentcultures). ^(3,4)Hyaluronan synthase (HAS) and hyaluronidase (Hyal)expression as determined by real-time RT-PCR. Ct values were normalizedby GAPDH mRNA and the fold differences are relative to BxPC3 expression.

Example 6 Measurement of Tumor Cell Hyaluronan Concentration andRelationship to Anti-Tumor Activity of PEGPH20

In this example, the concentration of hyaluronan in different tumor celllines was assessed by immunohistochemical analysis and compared to theability of PEGPH20 to inhibit tumor growth of xenograft tumors generatedfrom the tumor cell lines and tumor colony growth of an HA rich tumorcell line.

A. Comparison of HA Content with PEGPH20 Efficacy in Various XenograftTumor Models

1. Xenograft Tumor Models

Fourteen tumor cell line-derived xenograft tumors were generated fromthe following tumor cell lines: DU-145 human prostate carcinoma (ATCCHTB-81 mock transfected with empty pLXRN plasmid, see Example 5.A.1),DU-145 HAS2 (see Example 5.A.1), MDA MB 231 human breast adenocarcinoma(ATCC HTB-26), MDA-MB-231\Luc (D3H2LN, Caliper Life Sciences), MDA MB231 Luc/HAS2 (see Example 5.A.1), SKOV3 human ovarian carcinoma (ATCCHTB-77), AsPc-1 human pancreatic adenocarcinoma (ATCC CRL-1682), MIAPaCa 2 human pancreatic carcinoma (ATCC CRL-1420), 4T1 mouse breasttumor (ATCC CRL-2539), BxPC-3 human pancreatic adenocarcinoma (ATCCCRL-1687), Mat-Lylu rat malignant prostate carcinoma (ATCC JHU-92), andPC-3 human prostate adenocarcinoma (ATCC CRL-1435). Tumor cell lineswere maintained as described in Example 5. LUM 697 and LUM 330 humantumor explants were obtained from Crown Bioscience, Beijing China.

Six to eight week old nu/nu (Ncr) athymic nude mice (Taconic) or Balb/c(Harlan) or Balb/c nude mice (Shanghai Laboratory Animal Center, CAS(SLACCAS); see Example 7) were housed in micro-isolator cages, in anenvironment-controlled room on a 12-hour light/12-hour dark cycle, andreceived sterile food and water ad libitum. All animal studies wereconducted in accordance with approved IACUC protocols.

For generation of tumors, mice were inoculated with tumor cellsperitibially (intramuscular injection adjacent to the right tibiaperiosteum), subcutaneously (s.c., right hind leg), or in mammary fatpad according to Table 6 below.

TABLE 6 Animal Tumor Models Tumor Type Animal Inoculation Models(Source) Mice Source Site Cell Number Du145 Mock Prostate Ca. (H) Ncrnu/nu; female Taconic peritibially 5 × 10⁶/0.05 mL MDA MB 231 Breast Ca.(H) Ncr nu/nu; female Taconic peritibially 1 × 1 × 1 mm³ tissue cubeSKOV3 Ovarian Ca. (H) Ncr nu/nu; female Taconic s.c. 1 × 10⁶/0.1 mL MDAMB 231Luc Breast Ca. (H) Ncr nu/nu; female Taconic peritibially 1 ×10⁶/0.1 mL MDA MB 231 Breast Ca. (H) Ncr nu/nu; female Taconicperitibially 1 × 10⁶/0.1 mL Luc/HAS2 AsPc-1 Pancreatic Ca. (H) Ncrnu/nu; female Taconic s.c. 1 × 10⁷/0.1 mL MIA PaCa 2 Pancreatic Ca. (H)Ncr nu/nu; female Taconic s.c. 1 × 10⁷/0.1 mL 4T1 Breast Ca. (M) BALB/c;female Harlan mammary fat 5.0 × 10⁵/0.05 mL pad BxPC3 Pancreatic Ca. (H)Ncr nu/nu; female Taconic s.c. 1 × 10⁷/0.1 mL MatLylu Prostate Ca. (R)Ncr nu/nu; male Taconic peritibially 2.0 × 10⁵ cells/0.04 mL PC3Prostate Ca. (H) Ncr nu/nu; male Taconic peritibially 1 × 10⁶/0.05 mLDU145 HAS2 Prostate Ca. (H) Ncr nu/nu; male Taconic peritibially 5 ×10⁶/0.05 mL LUM858 Lung Ca. (H) BALB/c nude SLACCAS s.c. 3 × 3 × 3 mm³cube LUM 697 Lung Ca. (H) BALB/c nude SLACCAS s.c. 3 × 3 × 3 mm³ cubeLUM 330 Lung Ca. (H) BALB/c nude SLACCAS s.c. 3 × 3 × 3 mm³ cube H:Human; M: Mouse; R: Rat

For peritibial tumors, tumor volumes were determined using VisualSonicsVevo 770 high-resolution ultrasound. For subcutaneous and mammary fatpad tumors, tumor volumes were calculated by caliper measurement of thelength (L) and width (W) of the solid tumor masses. Tumor volume (TV)was calculated as: (L×W²)/2. Animals were selected for PEGPH20 treatmentwhen tumor volumes reached ˜400-500 mm³. Animals were then randomizedinto treatment and control groups (n≧6 mice/group).

Treatment with PEGPH20 and analysis of tumor growth inhibition (TGI)were performed as described (Thompson et al.). Mice were treated withvehicle (10 mM Histidine, pH 6.5, 130 mM NaCl) or PEGPH20 for 2-3 weeksaccording to the schedule shown in Table 7. Tumor volumes were measuredtwice weekly. When tumor size exceeded 2,000 mm³, animals were removedfrom the study and humanely euthanized.

TABLE 7 PEGPH20 Treatment and Tumor Growth Inhibition PEGPH20 PEGPH20amount per dose 0.1 mL dose Dosing No. of TGI Animal Models (mg/kg)(i.v.) Frequency Doses (day X) Number Du145 Mock 15 ~10,000 U  twiceweekly 6 0.7% (d18)  8 MDA MB 231 4.5 ~3,000 U twice weekly 6  0% (d17)5 SKOV3 5 ~3,500 U weekly 3  0% (d14) 5 MDA MB 4.5 ~3,000 U twice weekly5 23% (d14) 10 231Luc MDA MB 231 4.5 ~3,000 U twice weekly 5 43% (d14)10 Luc/HAS2 AsPc-1 4.5 ~3,000 U twice weekly 4 18% (d11) 9 MIA PaCa 24.5 ~3,000 U twice weekly 4 24% (d15) 9 4T1 3.9 ~3,000 U twice weekly 661% (d14) 5 BxPC3 4.5 ~3,000 U twice weekly 4 45% (d25) 7 MatLylu 3.9~3,000 U q2d 5 34% (d9)  5 PC3 15 ~10,000 U  twice weekly 6 65% (d18) 6DU145 HAS2 15 ~10,000 U  twice weekly 6 50% (d18) 8 LUM858 (see 4.5~3,000 U twice weekly 5 16% (d14) 10 Ex. 7) LUM 697 (see 4.5 ~3,000 Utwice weekly 5 97% (d14) 10 Ex. 7) LUM 330 (see 4.5 ~3,000 U twiceweekly 5 44% (d16) 10 Ex. 7)

The TGI for each tumor model was calculated based on the volume from thestudy termination day as indicated in Table 7. Percent Tumor GrowthInhibition (TGI) for each respective tumor model was calculated usingthe following equation:

% TGI=[1−(T _(n) −T ₀)÷(C _(n) −C ₀)]×100%

where “T_(n)” is the average tumor volume for the treatment group(animals receiving PEGylated rHuPH20) at day “n” after the final dose ofPEGylated rHuPH20; “T₀” is the average tumor volume in that treatmentgroup at day 0, before treatment; “C_(n)” is the average tumor volumefor the corresponding control group at day “n”; and “C₀” is the averagetumor volume in the control group at day 0, before treatment.Statistical analysis of tumor volumes between the control and treatmentgroups was performed using a one-way ANOVA test with P value of P≦0.05defined as statistically significant.

2. Histochemistry Staining of HA in Tumor Tissue and Semi-Quantificationof HA Content

At the termination of the tumor growth inhibition study, each of thefourteen xenograft tumors generated were analyzed for HA content byhistochemistry using biotinylated hyaluronan binding protein (B-HABP) asa probe for HA detection and digital quantification.

Tumor tissues were harvested, fixed in 10% neutral buffered formalinsolution (NBF), embedded in paraffin, and cut into 5 μm sections. Forhistochemical analysis, the sections were deparaffinized and rehydrated.Endogenous peroxidases were blocked with peroxo-block solution(Invitrogen, CA, USA) for 2 minutes. Non-specific staining was blockedusing 2% BSA in 2% normal goat serum PBS for 1 hour at room temperature(RT) prior to incubation with 2.5 μg/ml biotinylated HA-binding protein(B-HABP, Catalog No. 400763, Seikagaku, Tokyo, Japan) for 1 hour at 37°C. To confirm specificity of staining, a subset of sections werepre-treated with rHuPH20 (1000 U/mL) in PIPES buffer (25 mM PIPES, 70 mMNaCl, 0.1% BSA, pH 5.5) at 37° C. for 1 h before addition of B-HABP.After washing to remove the primary reagent, samples were incubated withstreptavidin-horseradish peroxidase solution (BD Pharmingen, Catalog No.550946) for 30 minutes at RT and detected with 3,3′-diaminobenzidine(DAB; Dako, Catalog No. K3467). Sections were then counterstained inGill's hematoxylin (Vector Labs, Catalog No. H-3401), dehydrated andmounted in Cytoseal 60 medium (American MasterTech).

An Aperio T2 Scanscope (Aperio) was used to generate high-resolutionimages of tissue sections. Images were quantitatively analyzed withAperio Spectrum software using a pixel count algorithm for brown color(HA) count. The tissue core in the sections with less than 10% of tumorcells or more than 50% of necrotic tissue was excluded for theevaluation. PC3 (HA⁺³) xenograft tumor tissues were used as a positivecontrol. A ratio of strong positive (brown) stain area to the sum oftotal stained area was calculated and scored as +3, +2, +1, or 0 whenthe ratio was more than 25%, 10-25%, less than 10%, or 0, respectively.

Spearman's rank correlation coefficient was used to evaluate therelationship between HA expression and response to PEGPH20 treatment.

3. Results: Comparison of Tumor HA Content and Tumor Growth InhibitionFollowing PEGPH20 Treatment

The results presented in Table 8 compare the level of HA measured intissue sections taken from the xenograft tumor and the percentage tumorgrowth inhibition (TGI) by PEGPH20. The results are from tumors treatedwith at least 1 mg/kg PEGPH20. Doses higher than 1 mg/kg did notincrease tumor growth inhibition. In the PC-3 (HA⁺³) and BxPC3 (HA⁺²)animal models, no significant increase in efficacy was observed at dosesgreater than 10 μg/kg and 100 μg/kg, respectively.

Despite the diversity of tumor cell types (human, murine and ratorigin), there was a significant correlation (P<0.001, Spearman's r=8,n=14) between increasing B-HABP-mediated HA staining intensity and invivo antitumor activity of PEGPH20 (Table 8).

TABLE 8 Tumor HA staining intensity and corresponding PEGPH20-mediatedgrowth inhibition. Tumor Type HA positive TGI Models (Source) Pixels (%)(%) DU145 Mock Prostate Ca. (H) 3.50 0 MDA MB 231 Breast Ca. (H) 4.59 0SKOV3 Ovarian Ca. (H) 4.87 0 MDA MB 231/Luc Breast Ca. (H) 6.97 23 MDAMB 231/Luc/HAS2 Breast Ca. (H) 11.18 43 AsPc-1 Pancreatic Ca. (H) 15.1518 MIA PaCa 2 Pancreatic Ca. (H) 17.08 24 LUM330 (see Ex. 7) Lung Ca.(H) 17.70 44 4T1 Breast Ca. (M) 19.45 45 BxPC3 Pancreatic Ca. (H) 20.5061 MatLylu Prostate Ca. (R) 24.00 34 PC3 Prostate Ca. (H) 27.27 65 DU145HAS2 Prostate Ca. (H) 28.85 50 LUM697 (see Ex. 7) Lung Ca. (H) 32.73 97H: Human; M: Mouse; R: RatB. Comparison of HA Content with PEGPH20 Efficacy in a Xenograft TumorModel of HAS2 Overexpression

The effect of increasing HA production in a tumor cell on increasing thesensitivity of tumors to treatment with PEGPH20 was further examined intumor xenografts that express exogenous hyaluronan synthase 2 (HAS2). Asshown in Example 5, HA production by the DU-145 tumor cell line could beincreased by transduction of the cells with a gene encoding HAS2, whichled to enhanced pericellular matrix formation in vitro. Additionally,the DU142-HAS2 displayed increased HA staining and increased tumorinhibition by PEGPH20 in the xenograft models described above. In thisExample, the efficacy of PEGPG20 treatment over time was compared in theDU-145 versus DU-145-HAS2 xenografts.

The mouse xenograft models were prepared as described above in Example6A. Briefly, mice were inoculated with either DU-145/vector controls orDU-145/HAS2 cells as indicated in Table 6. When the tumors reachedapproximately 500 mm³ in size, the mice were divided into treatmentgroups (n=8) and treated with vehicle alone or PEGPH20. For the PEGPH20treatment, the mice were injected via tail vein at a dose of 4.5 mg/kgtwice weekly for 3 weeks. Tumor volume was monitored by calipermeasurement as described above. The xenograft tumors were analyzed forHA content by histochemistry using biotinylated hyaluronan bindingprotein (B-HABP), as described above, 24 hours after the last treatmentwith PEGPH20.

The HAS2-overexpressing DU-145 prostate tumor xenograft grew moreaggressively in nude mice than the parental cell line transfected withempty vector (DU-145 Mock), similar to previous reports (Table 7)(Thompson et al. (2010)). PEGPH20 inhibited tumor growth in DU-145-HAS2tumors (TGI=50%, P<0.001, n=8), but not in DU-145 vector control tumors(TGI=0.7%, P>0.05, n=8). In addition, histochemistry staining withB-HABP of PEGPH20-treated tumors showed HA removal in tumor samplescompared to control tumors. These data suggest that accumulation of HAin the ECM facilitates tumor development, and that enhancedtumor-associated HA accumulation is associated with the anti-tumoractivity of PEGPH20.

C. Dose Related Effects of PEGPH20 Treatment in Hyaluronan-Rich Tumors

In this experiment, the dose dependent effect of PEGPH20 on tumor growthinhibition of HA-rich tumors was examined. Mouse xenograft models wereprepared as described above in Example 6A. Briefly, mice were inoculatedwith either BxPC-3 human pancreatic adenocarcinoma (ATCC CRL-1687) orPC-3 human prostate adenocarcinoma (ATCC CRL-1435) cells according toTable 6. When the tumors reached approximately 500 mm³ in size, the micewere divided into treatment groups (n=10) and treated with vehicle aloneor PEGPH20. For the PEGPH20 treatment, the mice were systemicallyinjected tail vein at a dose of 0.01, 0.1, 1, 4.5 and 15 mg/kg (350,3,500, 35,000, 157,500 and 500,000 U/kg, respectively) twice weekly for2 weeks. Tumor volume was monitored by caliper measurement as describedabove.

It was observed that the maximum effective dose of PEGPH20 is below 1mg/kg. Significant tumor inhibition was observed for all doses ofPEGPH20 in the PC-3 xenograft model (P<0.001 for 0.1, 1, 4.5, 15 mg/kgdoses; P<0.01 for the 0.01 mg/kg dose compared to vehicle) and for alldoses greater than 0.01 in the BxPC-3 xenograft model (P<0.001 for 0.1,1, 4.5, 15 mg/kg doses compared to vehicle). No significant increase inefficacy was observed at doses greater than 0.01 μg/kg (PC3, HA⁺³) or0.1 mg/kg (BxPC3, HA⁺²)

D. Effect of PEGylated rHuPH20 on Colony Growth of Hyaluronan-Rich TumorCells In Vitro

To determine whether PEGPH20 can inhibit anchorage-independent growthand proliferation of hyaluronan-rich prostate tumor cells (PC3) invitro, a three-dimensional clonogenic assay was performed on cells. PC3cells, at approximately 80% confluency, were trypsinized, harvested, andwashed once in completed medium. Cell density was adjusted to 8×10⁴/mLcells and suspended in Matrigel® (BD Biosciences, San Jose, Calif.) onice. 0.025 mL of this cell/Matrigel® mixture were seeded onto a 48 wellcell culture plate that had been pre-coated with Matrigel® at 0.1 mL perwell, and solidified at 37° C. for 1 hour. For continuous exposure, over17 days, to control API buffer and various concentrations of PEGPH20,0.6 mL/well of completed medium containing API buffer, 1, 3, 10 and 100U/mL of PEGPH20 were added to the top of the appropriate well. The wellswere incubated at 37° C., in a humidified atmosphere with 5% CO₂ in airfor 17 days, fresh treatment medium, including the appropriateconcentration of enzyme, where appropriate, was replaced every 3-4 daysduring the 17 day period.

On day 17, growth of colonies was assessed by capturing images with aNikon Eclipse TE2000U inverted microscope coupled to an Insight FireWiredigital camera (Diagnostic Instruments, Michigan). The colony number anddiameter of each colony in μm were measured using ImageJ software (opensource software, a publicly available program for display and analysisof images, for calculating area and pixel value) and coupled calibrationfunction (colony volumes were calculated using colony diameter and usingthe formula: 4/3πr³.

Average colony volume of wells for each condition were determined andthe effects of PEGPH20 on colony volume assessed by comparing theaverage colony volume in the control sample (API (active pharmaceuticalingredient) buffer (10 mM Hepes and 130 mM NaCl, pH 7.0) without enzyme)to the samples that were incubated in the presence of PEGylated rHuPH20Inhibitory ratios were calculated using the formula:

(mean volume of control−mean volume of treated)/(mean volume ofcontrol)*100.

PEGylated rHuPH20 induced a dose-dependent inhibition of growth,evidenced by lower colony volume compared with control. Based oninhibitory ratios calculated using the above formula, the culturesincubated in the presence of PEGPH20 at 1, 3, 10, and 100 U/mL exhibitedan average reduction in colony volume of 39%, 67%, 73%, and 75%respectively (p<0.01 for the 3 U and 10 U samples; p<0.001 for the 100 Usamples; n=6), compared to cultures incubated with control buffer.Statistical differences were analyzed using the Mann-Whitney Test.

The IC₅₀ of PEGPH20 in reducing colony volume, determined using theGraphpad Prism®4 program (GraphPad Software, Inc., La Jolla, Calif.),was approximately 1.67 U/mL. The average number of colonies was10.17±1.56 per well in vehicle-treated (control) cultures and 11.50±0.89per well in the cultures treated with PEGPH20 100 U/mL. The differencein colony number was not significant between the control and the 100U/mL cultures (n=6, p>0.05). These results indicate that PEGPH20 caninhibit proliferation and/or survival of hyaluronan rich cancer cells.

In an independent experiment, PC3 cells were seeded in reconstitutedbasement membrane (Matrigel) as described above and continuously exposedto vehicle or 0.1, 1, 10, 100, and 1000 U/mL of PEGPH20 for 19 days.Images were then digitally captured and colony volume was assessed usingthe ImageJ program. Inhibition of colony volume compared to control was22, 45, 63, 73 and 74%, respectively (P<0.01 for 1 U/ml, P<0.001 for 10U/mL and above compared to vehicle; n=3).

Example 7 Assessment of HA as a Biomarker for Predicting Response ofHuman NSCLC Tumors to PEGPH20

a. Expression of HA in NSCLC Patient Biopsies

Previous work has shown that elevated accumulation of HA occurs innon-small cell lung cancer (NSCLC) (Hernandez J R, et al. (1995) Int JBiol Markers 10: 149-155 and Pirinen R, et al. (2001) Int J Cancer 95:12-17). By contrast, NSCLC-derived cell lines exhibit low levelssuggesting the NSCLC cells lines lose HA expression during passaging invitro. Thus, HA expression in primary tumor biopsies was examined.

A tissue microarray (TMA) panel of 190 NSCLC biopsies (US Biomax, Inc.)

were examined for histotype and HA accumulation. HA content wasdetermined by B-HABP histochemistry staining as described in Example 6.Samples were scored as +3, +2, +1 or 0 when the ratio of strong positive(brown) stain area to the sum of total stained area was more than 25%,10-25%, less than 10% or 0, respectively. In this panel, adenocarcinoma(ADC), squamous cell carcinoma (SCC), and large cell carcinoma (LCC)cell types were observed at frequencies of 32%, 51%, and 3%,respectively, classified based on pathology diagnosis provided by USBiomax (Table 9). Other unidentified subtypes comprised about 11% of the190 samples examined.

Analysis of tumor-associated HA accumulation showed that all histotypeshave subsets of cells which express the HA⁺³ high HA phenotype, with anoverall rate of approximately 27% (Table 9). In particular, 40% of SCCcases displayed the HA phenotype, while 11% of ADC and 33% of LCC caseswere scored as HA⁺³. 34% of SCC cases displayed the HA⁺² phenotype,while 48% of ADC and 50% of LCC cases were scored as HA⁺². 25% of SCCcases displayed the HA⁺¹ phenotype, while 36% of ADC and 17% of LCCcases were scored as HA⁺². In this dataset, none of the normal lungtissue samples expressed the HA⁺³ phenotype, although detectable HA wasobserved in most samples of normal lung tissue.

TABLE 9 Distribution of HA expression in human lung cancer samples HApositive incidence N (%) HA score¹ ADC² SCC3² LCC⁴ Other Normal  0  3(4.7) 0 (0) 0 (0) 1 (3) 0 (0) +1 23 (36) 24 (25) 1 (17) 11 (48)  9 (43)+2 31 (48) 33 (34) 3 (50) 9 (39) 12 (57) +3  7 (11) 40 (41) 2 (33) 2(8.7) 0 (0) Total 62 (32) 99 (52) 6 (3) 23 (12)  21 (100) ¹HA scoreswere defined based on % of positive HA staining intensity ²ADC:Adenocarcinoma ³ SCC: Squamous Cell Carcinoma ⁴LCC: Large Cell Carcinoma

B. Expression of HA in NSCLC Patient Explants and Prediction of PEGPH20Efficacy

In order to prospectively test the relationship between HAoverexpression and antitumor response of NSCLC to PEGPH20-mediated HAdepletion, human NSCLC patient tumor explants representing differentdegrees of HA accumulation were selected and assessed for responsivenessto PEGPH20 treatment in a xenograft tumor model. Primary explantscharacterized for HA accumulation were used for this study becauseexplant models contain a more representative sampling of the geneticdiversity of intact tumors, and should retain aspects of nativetumor-like stroma.

Tumor biopsies were obtained from sixteen NSCLC patients, and weremaintained at a low passage number subcutaneously in nude mice (CrownBioscience, Beijing, China). The NSCLC tumor explants were screened forHA accumulation in explant tissues from passages 1-4 and were assignedan HA phenotype (i.e., +1, +2 or +3) by B-HABP histochemistry stainingas described above. Three squamous cell-type (SCC) explants wereprospectively selected for xenograft transplantation, representing theHA⁺³ (LUM697), HA⁺² (LUM330), and HA⁺¹ (LUM858) phenotypes.

When the seed tumors for the selected tumor explants reached 500-700 mm³in size, the mice were sacrificed and the tumors were extracted andminced into 3×3×3 mm³ fragments. One fragment for each tumor wassubcutaneously implanted into the right rear flank of a female Balb/cnude mouse (n=10 for each group) as indicated in Table 6. Tumor volumeswere determined by caliper measurements of the greatest longitudinaldiameter (length (L)) and the greatest transverse diameter (width (W))and estimated using the calculation of (L×W²)/2. When the average tumorsize reached 500 mm³ (range 300-600 mm³), the animals were randomizedinto two groups. For therapy, animals were treated with vehicle orPEGPH20 at 4.5 mg/kg twice weekly for 5 doses as shown in Table 7 above.The percentage tumor growth inhibition (% TGI) and statistical analysiswere performed as described Example 6. The rank-order of HA phenotype(i.e., +1, +2 or +3) as determined by histochemistry was found topredict the degree of tumor growth inhibition by PEGPH20 (Table 9). Forexample, the percentage growth inhibition was 97% for LUM697 (HA⁺³), 44%for LUM330 (HA⁺²), and 16% for LUM858 (HA⁺¹). In addition, tumorregression was observed in the LUM697 (HA⁺³) tumor explant group, butnot the LUM330 (HA⁺²) and LUM858 (HA⁺¹) groups: 4 of 10 animals withLUM697 (HA⁺³) tumors had decreased tumor burden compared to pretherapy.

Example 8 Effect of PEGPH20 Treatment on Xenograft Tumor Cell DNASynthesis and the Tumor Microenvironment (TME) A. Effect of HA Depletionon Tumor Cell DNA Synthesis

To test whether HA depletion has antiproliferative effects on tumorcells in vivo, PC-3 (HA⁺³) tumor xenografts treated with PEGPH20 wereexamined for levels of DNA synthesis.

Six to eight week old nu/nu (Ncr) athymic nude mice intraperitibiallyimplanted with PC-3 tumor cells as described in Example 6 (1×10⁶ cell in0.05 mL per mouse). Tumor volume was monitored by caliper measurement.When the tumors reached ˜400 mm³, the mice were treated with vehicle orPEGPH20 (1 mg/kg (35,000 U/kg) or 4.5 mg/kg (157,500 U/kg); about 700U/dose or 3150 U/dose based on 20 g mouse body weight), 100 μL via tailvein injection twice weekly for two weeks. The 24 hours before studytermination, the mice were administered 10 mg/kg BrdU (0.2 mL)(Invitrogen, Cat#00-0103) intraperitoneally. Tumors were excised fromthe mice, fixed in 10% buffered formalin and embedded in paraffin.Tissues were cut into 5 μm sections, and cell proliferation was assessedafter staining with an anti-BrdU antibody (BrdU Staining Kit;Invitrogen, Cat#93-3943) according to the manufacturer's instructions.

Animals treated with PEGPH20 were compared to vehicle-treated animals. A58.3% reduction in synthetically active nuclei was observed in thePEGPH20 treated tumors compared to vehicle-treated tumors (percent BrdUpositive nuclei was reduced from 4.8% to 2%). This result parallels theobserved growth inhibition of prostate PC3 (HA⁺³) or pancreatic BxPC3(HA⁺²) xenografts as a result of PEGPH20 treatment (˜50% TGI at doses of1 mg per kg or more) (see Example 6).

B. Effect of HA Depletion on Expression of Tumor MicroenvironmentAssociated Proteins

Previous studies have shown that treatment of HA⁺³ tumors with PEGPH20has a dramatic effect on tumor interstitial fluid pressure (IFP), andtherefore on the fluid pressure differential between the tumor and itsexternal environment (see Thompson et al. (2010)). Physical changes inthe TME can have an impact gene expression (Shieh AC (2011) Ann BiomedEng 39:1379-1389). In order to test whether removal of HA has an impacton turnover or expression of TME proteins, expression of TME proteins,such as murine collagen I (Col1α1), murine collagen V (Col5α1), andtenascin C (TNC), which are found in the actively remodeling matrix,were examined.

1. Localization and Semi-Quantification of Collagen in Tumor Tissue

Tumor tissues with adjacent skin from PC-3 tumors generated in Example8A were fixed in 10% neutral buffered formalin for 48 hours, processedusing a tissue processor (TISSUE-TEKVIP, Sakura Finetek, CA) andembedded in paraffin block. The paraffin-embedded tissue samples werecut into 5 μm sections, dewaxed, and rehydrated in deionized water.Antigen retrieval was processed by heating slides in EDTA buffer at pH8.0, 100° C. for 25 min. Slides were rinsed in PBS-T, blocked with 2%normal goat serum in 2% PBS/BSA for 30 min, followed by incubation withrabbit polyclonal anti-collagen type 1 antibody (1:200, Abcam,Cat#ab34710) for 2 hours at room temperature. The sections were thenincubated in Texas red tagged goat anti-rabbit IgG (1:200, VectorLaboratories, Cat# F1-1000) for 1 hour at room temperature, and counterstained and mounted with ProLong® Gold antifade reagent with DAPI(Invitrogen, CA). Micrographs were captured under a Zeiss Axioskopmicroscope coupled with RT3 camera (Diagnostic Instruments, MI). Random5 fields from each section were analyzed for collagen-positive intensityusing Image-Pro plus program.

2. cDNA Arrays Analysis of Gene Expression in PC3 Xenograft Tumor Tissue

NCR nu/nu mice bearing PC3 tumors were generated and treated withvehicle or PEGPH20 as described in Example 6A. Animals were euthanized 8and 48 hours post-treatment with vehicle or PEGPH20. Tumor tissues wereexcised in sterile conditions and snap frozen in liquid nitrogen. TotalRNA was isolated from frozen tissue according to Asuragen's standardoperating procedures. The purity and quantity of total RNA samples weredetermined by absorbance readings at 260 and 280 nm using a NanoDropND-1000 UV spectrophotometer. The integrity of total RNA was qualifiedby Agilent Bioanalyzer 2100 microfluidic electrophoresis. Samples formRNA profiling studies were processed by Asuragen, Inc. using AffymetrixMouse 430 2.0 and Human U133 plus 2.0 arrays.

3. Results

Tumor-specific reduction of Col1α1 in the PC-3 tumors was observedfollowing depletion of HA by treatment with PEGPH20. 80% reduction inCol1α1 staining compared to vehicle treated tumors was observed (P<0.05t test). Col1α1 staining in skin from PEGPH20 treated mice, however,remained stable. In addition, decreased levels of murine (stromal) mRNAsfor Col1α1, Col5α1, and TNC were observed as measured by mRNA expressionarray analysis. TNC mRNA was most significantly impacted (66% decrease),followed by Col1α1 (53% decrease) and Col5α1 (45% decrease). Theseresults suggest that depletion of HA results in significant changes inthe expression of proteins within the TME.

Example 9 Generation of TSG-6 Link Module IgG Fc Fusion Protein

A fusion protein, TSG-6-LM-Fc, containing the link module of TSG-6 andthe Fc domain of IgG was generated. A mutant fusion proteinTSG-6-LM-Fc/ΔHep in which the heparin binding region of the TSG-6 linkmodule was mutated, also was generated.

A. Vector Construction of Recombinant Human TSG-6 Link Module FusionProteins

DNA de novo synthesis (GenScript, NJ) was employed to generate nucleicacid encoding the TSG-6-LM-Fc fusion protein. The nucleic acid containsa DNA encoding a human immunoglobulin light chain kappa (κ) leadersignal peptide sequence (SEQ ID NO:210), a 669 bp-long cDNA fragment ofhuman IgG1 heavy chain (GI No. 5031409; SEQ ID NO: 203, encoding thepeptide sequence set forth in SEQ ID NO:204) and a 285 bp-long cDNAfragment of human TSG-6 link module region (SEQ ID NO:216, encoding thepeptide sequence set forth in SEQ ID NO:207, which corresponds to aminoacid positions 35 to 129 of the TSG-6 preprotein, GI No. 315139000, setforth in SEQ ID NO:205 (mRNA) and SEQ ID NO:206 (protein)). The humanIgG1 heavy chain and human TSG-6 link module regions were connected witha 6 bp AgeI restriction enzyme cleavage site and a 12 bp sequence,GACAAAACTCAC (SEQ ID NO: 208), encoding four additional amino acids(DKTH; SEQ ID NO: 209) originally published as part of the IgG1 Fcsequence (Nucleic Acids Research, 1982, Vol. 10, p4041). Two uniquerestriction enzyme cleavage sites, NheI at 5′ end and BamHI at 3′end,were synthesized flanking the fusion protein sequence. The synthesizedfragment has a sequence set forth in SEQ ID NO:217. The fragment wascodon optimized for improved protein expression and synthesized by denovo DNA synthesis. The codon optimized fragment has a sequence setforth in SEQ ID NO:211. The protein sequence for the encoded TSG-6-LM-Fcfusion protein is set forth in SEQ ID NO: 212.

The synthesized codon optimized fragment was inserted via NheI and BamHIcleavage sites into the pHZ24 IRES bicistronic mammalian expressionvector (SEQ ID NO: 52) using well-known recombinant DNA procedures(restriction enzyme and ligation reagents obtained from New EnglandBiolabs, Ipswich, Mass.) to generate pHZ24-TSG-6-LM-Fc construct (SEQ IDNO:213). Recombinant protein expression in this vector is driven by aCMV promoter.

In order to enhance the hyaluronan (HA) binding specificity and reducebinding to other GAG chains, a construct encoding a mutant fusionprotein, TSG-6-LM-Fc/ΔHep, that contains 3 lysine to alanine mutationsat amino acid positions 55, 69, 76 of the TSG-6 link module wasconstructed. The mutations reduce the heparin binding activity of theTSG-6 link module, while not affecting the HA binding activity (seeMahoney D J et al. (2005) J Biol Chem. 280:27044-27055, which reports10-fold lower heparin binding activity for the triple mutant;K20A/K34A/K41A in the heparin binding site). TSG-6-LM-Fc/ΔHep wasgenerated by mutagenesis of the nucleic acid fragment encoding theTSG-6-LM-Fc fusion protein and insertion into the pHZ24 IRES vector togenerate pHZ24-TSG-6-LM-Fc/ΔHep (SEQ ID NO:218). The sequence of theTSG-6-LM-Fc/ΔHep fragment is set forth in SEQ ID NO: 214, which encodesthe TSG-6-LM-Fc/ΔHep fusion protein set forth in SEQ ID NO: 215.

B. Recombinant Protein Expression and Purification

FreeStyle CHO-S suspension cells (Invitrogen) were employed forexpression of TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep fusion proteins. TheFreeStyle CHO-S suspension cell line was maintained in CHO-S CD culturemedium (Invitrogen) prior to transfection. For preparation of the cellsfor transfection and recombinant protein expression, FreeStyle CHO-Scells were cultured in FreeStyle CHO Expression Medium (Invitrogen)supplemented with 8 mM L-glutamine in shake flasks at 37° C. in ahumidified atmosphere of 8% CO2 in air on an orbital shaker platformrotating at 125 rpm with loosened caps of flasks to allow for aeration.

Transient transfection of suspension cells was performed according tothe manufacturer's instructions. Briefly, cells were split at a density6×10⁵/ml 24 hours before transfection, and transfected using FreeStyleMax lipid with a DNA/lipid ratio at 1:1. After 96 hourspost-transfection, cells were harvested at 4,000 g for 20 min, andsupernatants were collected. A time course analysis of proteinexpression level during the post-transient transfection revealed thatthe protein expression level reached a plateau after 96 hours posttransfection. Thus, the recombinant protein was collected at 96 hourpost-transfection.

The expressed TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep fusion proteins in thecollected supernatants were affinity purified by Protein A resins(Bio-Rad, Hercules, Calif.) according to the manufacturer'sinstructions. Briefly, the collected supernatants were adjusted to pH7.4, 0.15 M NaCl with 1 M Tris-HCl, pH 7.4 (Teknova Catalog No. T1074)and 5M NaCl (Sigma) and diluted with binding buffer 3 fold before loadedonto a Protein A column. The eluted product was immediately neutralizedwith 1M Tris-HCl, pH 8.5, and dialyzed against Phosphate-BalancedSolution (PBS, 137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.46 mM KH₂PO₄,and pH 7.4) at 4° C., and stored at −20° C. The yield of the purifiedproteins from the supernatants through a single step Protein A affinitycolumn was between 3 to 5 mg/liter.

C. SDS-PAGE and Western Blot Analysis of Expressed Recombinant Proteins

The purity, size and identity of the purified fusion protein weredetermined by SDS-PAGE 4-20% gradient gel under reducing andnon-reducing conditions and Western Blot analysis. 60 ng of purifiedprotein was used in the analysis. The size of the purified fusionproteins were about 40 kDa under reducing conditions and about 80 kDaunder non-reducing conditions, indicating the expressed proteins formhomodimers via disulfide bonds in hinge region of IgG Fc. The purity ofthe protein samples were greater than 95%. The purified proteins werestable in PBS for at least one month at 4° C. without any visibledegradation, or loss of binding activity.

The identity of the TSG-6 link module in TSG-6-LM-Fc andTSG-6-LM-Fc/ΔHep was assessed by Western blot with goat anti-human TSG-6IgG (R&D Systems, Inc., Minneapolis, Minn.) followed by rabbit anti-goatIgG-HRP (EMD, San Diego, Calif.). Recombinant full length human TSG-6protein (R&D Systems, Inc., Minneapolis, Minn.) was employed as apositive control. The pattern of detected proteins by Western blotanalysis under reducing and non-reducing conditions was the same as thatof SDS-PAGE analysis except for a small amount of upper bands observedunder the non-reducing condition, most likely representing tetramers ofthe recombinant proteins based on their molecular weight size.

The identity of the Fc portion in the purified recombinant proteins wasconfirmed by Western blot analysis with HRP-rabbit anti-human IgGFc(Jackson ImmunoResearch, West Grove, Pa.). The pattern of detectedproteins was the same as for the SDS-Page and anti-TSG-6 analyses,indicating that the purified proteins contain both TSG-6 link module(LM) as well as hIgGFc.

To analyze whether the proteins were glycosylated, the purified proteinswere treated with glycosidase PNGase F (0.5 units per ng protein), whichremoves the N-linked oligosaccharides from proteins, and analyzed bySDS-PAGE and Western blot. A 5 kDa difference of molecular weights ofproteins was observed between before and after treatment with PNGase F,indicating that the expressed proteins were glycosylated.

Example 10 Binding of TSG-6 to Hyaluronan and Heparin

Two formats were used to test the binding of both TSG-6-LM-Fc and itsmutant to HA and heparin. In one format, binding of TSG-6-LM-Fc andTSG-6-LM-Fc/ΔHep to immobilized HA or Heparin on a microplate wasemployed. In the second format, binding of biotinylated HA and heparinto immobilized recombinant TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep proteins ona microplate was employed.

A. Binding of Recombinant TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep toImmobilized HA and Heparin

Wild type and mutant TSG-6-LM Fc fusion homodimers were tested for theirHA binding and heparin binding activities using either HA orheparin-coated microplates. Briefly, hyaluronan with an average MW ofabout 1000 kDa (Lifecore, Chaska, Minn.) or Heparin with an average MWof 15 kDa (Calbiochem, San Diego, Calif.) at concentration of 100 μg/mlin 0.5 M sodium carbonate buffer, pH 9.6, was dispensed into 96-wellplates in duplicate, 100 μl/well, and incubated at 4° C. overnight.Plates were blocked with 1% BSA in PBS to reduce non-specific binding.

TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep purified protein samples were dilutedto give rise to concentration range from 0.31 to 40 ng/ml for binding toHA coated plate, 0.78 to 100 ng/ml for binding to heparin coated plate.For each sample, 100 μl per well in duplicate was added to themicroplate and incubated at room temperature for 1 hour. Plates werewashed PBS with 0.05% Tween 20, 5 times to remove unbound protein.Hyaluronan or heparin bound TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep weredetected with rabbit anti-human IgG Fc-HRP (Jackson ImmunoResearch, WestGrove, Pa.) followed by TMB (3,3′,5,5′-tetramethylbenzidine) substrate(KPL, Gaithersburg, Md.). The samples were incubated 60 minutes with therabbit anti-human IgG Fc-HRP antibody. After washing, bound HRP wasdetected with TMB solution over 10-15 minutes development time followedby addition of phosphoric acid reagent to stop color development.Absorbance was measured at OD450 using a Molecular Devices, Spectra M3spectrophotometer.

Both TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep displayed the same HA bindingactivity on the HA coated plate; and their titration curves of HAbinding activity were almost overlapped, indicating that the twoexpressed proteins bind HA with high affinity based on the EC₅₀ valuesfrom titration curves of HA binding. The triple mutation in the heparinbinding site has no effect on its HA binding. In contrast, the bindingof the two proteins to the heparin coated plate showed a significantdifference. The wild type TSG-6-LM-Fc bound heparin although withrelatively low binding activity compared to its binding to HA, whichcould be due to the size difference of the two GAG chains coated on theplates. The mutant TSG-6-LM-Fc protein exhibited about 10% of heparinbinding activity compared to that of wild type, which was consistentwith the reported result for the triple-mutated TSG-6-LM monomer(Mahoney D J et al. (2005)).

B. Binding of Biotinylated HA and Heparin to Immobilized RecombinantTSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep

The GAG binding properties of wild type TGS6-LM-Fc and TSG-6-LM-Fc/ΔHepwere further examined by coating microplates with the recombinantproteins and assessing their binding to biotinylated HA and biotinylatedheparin.

For preparation of the microplates, TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep ata concentration of 2 μg/ml in 1×PBS buffer was dispensed into 96-wellplates in duplicates, 100 μl/well, and incubated at 4° C. overnight.Plates were blocked with 1% BSA in PBS to reduce non-specific binding.

TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep purified protein samples were dilutedto give rise to concentration range from 0.31 to 40 ng/ml for binding toHA coated plate, 0.78 to 100 ng/ml for binding to heparin coated plate.100 μl per well for each sample in duplicate was added to the microplateand incubated at room temperature for 1 hour. Hyaluronan or heparinbound TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep were detected with anti-human IgGFc-HRP (Jackson ImmunoResearch, West Grove, Pa.) followed by TMB(3,3′,5,5′-tetramethylbenzidine) substrate (KPL, Gaithersburg, Md.).

For biotinylation of HA, the carboxyl groups on HA were used for theconjugation via hydrazide chemistry. Briefly, biotin-hydrazide wasdissolved in DMSO at a concentration of 25 mM, and added at a volumeratio of 6:100 into an HA solution, containing 1000 kDa or 150 kDamolecular weight HA (Lifecore Biomedical, LLC Chaska, Minn.) at 1 mg/mlin 0.1 M MES, pH 5.0. 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (ECD) and sulfo-N-Hydroxysuccinimide (sulfo-NHS) wereadded in the conjugation reaction to a concentration of 40 μM and 850μM, respectively, to mediate the conjugation of biotin-hydrazide and HA.The reaction was kept at 4° C. overnight while stirring. The excessamount of chemicals was removed from biotinylated HA by dialysis.Biotinylated heparin was purchased from EMD, San Diego (Catalog No.375054).

Biotinylated hyaluronan or heparin were diluted in PBS withconcentration range from 0.78 ng/ml to 100 ng/ml, dispensed 100 μl/well,and incubated at room temperature for 1 hour. Plates were washed withPBS with 0.05% Tween 20, 5 times to remove unbound protein. The boundbiotinylated hyaluronan and heparin were detected withanti-Streptavidin-HRP (Jackson ImmunoResearch, West Grove, Pa.) followedby TMB substrate (3,3′,5,5′-tetramethylbenzidine) substrate (KPL,Gaithersburg, Md.) as described above. Absorbance was measured at OD450.

The binding results observed were similar to the binding assay performedin Example 10A, which used immobilized HA and heparin and freeTSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep. There was no difference of bindingactivity of immobilized TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep to biotinylatedHA or in the binding titration curves between TSG-6-LM-Fc andTSG-6-LM-Fc/ΔHep, and a significant reduction in the binding of mutantTSG-6-LM-Fc/ΔHep to biotinylated heparin compared to that of wild typeprotein also was observed. Therefore, the HA and heparin bindingproperties of wild type TSG-6-LM-Fc and its mutant can be evaluated ineither GAG coated or recombinant protein coated format; and both formatsrevealed similar binding patterns.

C. Calculation of Binding Affinity of TSG-6-LM-Fc

The HA binding affinity of TSG-6-LM-Fc was measured using Bio-LayerInterferometry (BLI) technology via Octet QKe instrument (ForteBio,Menlo Park, Calif.). The full length TSG-6 recombinant protein (R&DSystems, Inc., Minneapolis, Minn.) was used as control. Briefly,biotinylated HA with an average molecular weight of 150 kDa wasimmobilized on streptavidin coated biosensors for 240 seconds.TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep was then associated with immobilized HAfor 180 seconds at different concentrations in PBS at pH 6.0 or pH 7.4,followed by dissociation of bound proteins in PBS at pH 6.0 or pH 7.4for 240 seconds. The results of binding kinetics were analyzed by thesoftware provided by the manufacturer. Results for the calculatedbinding affinity are provided in Table 10.

TABLE 10 Binding Affinity of TSG-6-LM-Fc Conc. Sample ID (nM) pH KD (M)kon (1/Ms) kdis (1/s) Full R{circumflex over ( )}2 TSG-6- 18.8 6.05.45E−09 2.46E+05 1.34E−03 0.970616 LM-Fc TSG-6- 6.25 6.0 5.45E−092.46E+05 1.34E−03 0.970616 LM-Fc TSG-6- 18.8 7.4 1.41E−08 4.44E+046.24E−04 0.986378 LM-Fc TSG-6- 6.25 7.4 1.41E−08 4.44E+04 6.24E−040.986378 LM-Fc

Example 11 Competitive Inhibition Assessment of TSG-6 Binding toHyaluronan and Heparin by Other Glycosaminoglycans

The HA and heparin GAG binding sites of the TSG-6 link module arelocated at different regions of the link module. In order to determinewhether the two binding sites would interfere with each other during theinteraction with TSG-6 link module or in the presence of other GAGchains, a competitive inhibition assay was performed to assess bindingof HA or heparin in the presence of other GAG chains.

HA and heparin coated 96-well microplates were prepared as described inExample 10A. TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep, at a concentration of 40ng/ml for the HA coated plates and 100 ng/ml for the Heparin coatedplates, were pre-incubated with four different GAG chains: HA (LifecoreBiomedical, LLC Chaska, Minn.), chondroitin sulfate A (EMD, San Diego,Calif., Catalog No. 230687) chondroitin sulfate C (EMD, San Diego,Calif., Catalog No. 2307) and heparin sulfate (EMD, San Diego, Calif.,Catalog No. 375095), at three different concentrations (0.11, 0.33, 1.0μg/ml) or without GAG chain as control at room temperature for 10minutes. The samples were then dispensed (100 μL) in duplicate into theHA and heparin coated 96-well microplates and incubated at roomtemperature for 1 hour. Plates were washed with PBS with 0.05% Tween 20,5 times, to remove unbound protein. Bound TSG-6-LM-Fc andTSG-6-LM-Fc/ΔHep were detected with anti-human IgG Fc-HRP (JacksonImmunoResearch, West Grove, Pa.) followed by TMB(3,3′,5,5′-tetramethylbenzidine) substrate (KPL, Gaithersburg, Md.) asdescribed above. Absorbance was measured at OD450.

For the HA coated plate, both TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep revealedsimilar competitive inhibition patterns. Binding of TSG-6-LM-Fc to theimmobilized HA was efficiently inhibited by pre-incubation of sameamount of protein with the different doses of free HA (approximately68%, 85%, and 93% inhibition for the 0.11, 0.33, 1.0 μg/ml doses,respectively), but was not affected by pre-incubation with differentdoses of free heparin or chondroitin sulfate C. Some inhibition ofTSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep was observed for pre-incubation withchondroitin sulfate A, though it was less than for HA (approximately23%, 43%, and 63% inhibition for the 0.11, 0.33, 1.0 μg/ml doses). Thus,an approximately 10 fold higher amount of chondroitin sulfate A wasneeded for inhibition. (In independent experiments up to 30-fold higheramount of chondroitin sulfate A was needed for inhibition compared toHA). Because TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep showed similar inhibitionwith pre-incubation with chondroitin sulfate A, it is likely that the HAbinding site in TSG-6 link module is responsible for the chondroitinsulfate A binding.

For the heparin coated plates, the binding of TSG-6-LM-Fc to heparin wasefficiently inhibited not only by pre-incubation with heparin, but alsoby pre-incubation with either HA or chondroitin sulfate A. This datashows that the binding of TSG-6-Fc-LM to HA could block its heparinbinding activity. As expected, mutant TSG-6-LM-Fc/ΔHep did not bindheparin and thus exhibited readings close to background for both controland pre-incubation samples.

This study demonstrates that binding of link module of TSG-6 to HA isnot affected by the presence of free heparin or preformed TSG-6 heparincomplex, while its binding to heparin is significantly inhibited by thepresence of free HA or preformed TSG-6 HA. Based on these observations,one can conclude that TSG-6-LM binds to HA and heparin simultaneously orbinding of TSG-6-LM to HA is stronger than its binding to heparin. HAand TSG-6-LM complex formation can cause protein conformation change orother arrangements of the protein that are not favorable for its bindingto heparin.

Example 12 Comparison of Glycosaminoglycan Binding Properties ofTSG-6-LM-Fc, TSG-6-LM-Fc/ΔHep and HABP

In this example, the specificity and binding activity of TSG-6-LM-Fc,TSG-6-LM-Fc/ΔHep and HA binding protein (HABP) to HA, heparin, and otherGAGs were compared. For this experiment, biotinylated-TSG-6-LM-Fc andbiotinylated-TSG-6-LM-Fc/ΔHep HA binding proteins were generated andcompared to commercially available biotinylated-HA binding protein(HABP) (Seikagaku, Tokyo, Japan) for their binding activity on GAG chaincoated plates.

A. Biotinylation of TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep

A random labeling approach was used to conjugate the biotin to primaryamine containing residues (Lys) in the protein directly withoutpre-incubation with free HA in order to protect HA binding sites. Forbiotinylation of TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep, direct conjugation ofthe primary amine active reagent NHS-PEG₄-Biotin (Thermo FisherScientific, Chicago, Ill.) was performed according to the manufacturer'sinstructions. 0.5 mg protein in PBS at concentration 1 mg/ml and 10 μlof 20 mM biotinylation reagent was used for the biotinylation reaction.The N-hydroxysuccinimide ester (NHS) group of NHS-PEG₄-Biotin reactsspecifically and efficiently with lysine and N-terminal amino groups atpH 7-9 to form stable amide bonds. The hydrophilic polyethylene glycol(PEG) spacer arm imparts water solubility that is transferred to thebiotinylated molecule, thus reducing aggregation of labeled proteinsstored in solution. The PEG spacer arm also gives the reagent a long andflexible connection to minimize steric hindrance involved with bindingto avidin molecules. Unreacted NHS-PEG₄-Biotin was removed with dialysisagainst 1×PBS and stored at −20° C.

For comparison, the TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep proteins also werebiotinylated using the oriented labeling approach, which conjugates thebiotin units to sugar chains on the proteins by oxidation ofpolysaccharide chain on the protein using NaIO₄ followed bybiotin-hydrazide. Briefly, 1 ml protein at a concentration of 1 mg/ml in0.1 M phosphate buffer, pH 7.2, was first oxidized by sodium periodate(NaIO₄) at a final concentration of 5 mg/ml, at 4° C. for 30 minutes.The reaction converts the two adjacent primary hydroxyl groups on sugarsto corresponding aldehyde reactive groups. The oxidized protein wasdialyzed against 0.1 M phosphate buffer, pH 7.2. The dialyzed proteinwas then mixed with 50 mM hydrazide-biotin prepared in DMSO at volumeratio 9 to 1 resulting in 5 mM hydrazide-biotin in the reaction andincubated at room temperature for 2 hours to form hydrazone bondsbetween aldehyde groups and hydrazide groups. The labeled protein wasdialyzed against 1×PBS and stored at −20° C.

After conjugation and removal of free biotin, the HA binding activity ofboth biotin-TSG-6-LM-Fc and biotin-TSG-6-LM-Fc/ΔHep were tested togetherwith non labeled corresponding proteins to examine if the labeling wouldcause reduced HA binding activity using the binding assay as describedin Example 10A using HA coated plates. No difference in HA bindingactivity was found between labeled vs non labeled proteins.

B. Binding of Biotinylated-TSG-6-LM-Fc, Biotinylated-TSG-6-LM-Fc/ΔHepand Biotinylated-HABP to GAGs

For preparation of the GAG coated microplates, HA, Heparin, chondroitinsulfate A, or chondroitin sulfate C, at a concentration of 100 μg/ml in0.5 M sodium carbonate buffer, were dispensed, 100 μl/well, into 96-wellplates in duplicate, and incubated at 4° C. overnight. Plates wereblocked with 1% BSA in PBS to reduce non-specific binding. The threebiotinylated proteins, biotinylated-TSG-6-LM-Fc,biotinylated-TSG-6-LM-Fc/ΔHep and biotinylated-HABP were diluted toconcentrations ranging from 0.05 to 100 ng/ml for binding to HA,chondroitin sulfate A, and chondroitin sulfate C coated plates, and 0.23to 500 ng/ml for binding to heparin coated plates. The diluted proteinsamples were dispensed onto the plates, 100 μl/well in duplicate, andincubated at room temperature for 1 hour. The proteins bound to the GAGcoated plates were detected with Streptavidin-HRP (JacksonImmunoResearch, West Grove, Pa.) followed by TMB(3,3′,5,5′-tetramethylbenzidine) substrate (KPL, Gaithersburg, Md.) asdescribed above. Absorbance was measured at OD450.

All three biotinylated GAG binding proteins exhibited strong HA bindingactivity on the HA coated plate. At 11.1 ng/ml protein concentration,which represented one dilution lower than maximal binding concentrations(i.e. 33.3 ng/ml and 100 ng/ml) for HA, binding ofbiotinylated-TSG-6-LM-Fc and biotinylated-TSG-6-LM-Fc/ΔHep to HA wasapproximately 14 fold over background, and B-HABP binding to HA wasapproximately 9 fold over background.

Both biotinylated-HABP and biotinylated-TSG-6-LM-Fc/ΔHep displayedlittle binding activity against the heparin coated plate.Biotinylated-wild type TSG-6-LM-Fc also showed negative in heparinbinding activity, suggesting that the random labeling approach withNHS-PEG₄-Biotin caused a loss of heparin binding activity. WhenTSG-6-LM-Fc was biotinylated by the oriented labeling approach asdescribed above, binding to heparin was restored and the proteinexhibited similar heparin binding activity as that of non-labeledTSG-6-LM-Fc. Thus, biotin modification of lysines in heparin site ofTSG-6-LM-Fc should abolish its heparin binding activity.

All three proteins exhibited no binding activity to chondroitin sulfateC coated plate, but demonstrated strong binding towards chondroitinsulfate A coated plate. The biotinylated-TSG-6-LM-Fc andbiotin-TSG-6-LM-Fc/ΔHep were observed to a few fold higher bindingactivity than that of biotin-HABP. At 11.1 ng/ml protein concentration,binding of biotinylated-TSG-6-LM-Fc and biotinylated-TSG-6-LM-Fc/ΔHep toHA was approximately 20 fold and 12 fold over background, respectivelyand B-HABP binding to HA was approximately 6 fold over background.Nonetheless, both TSG-6-LM-Fc and TSG-6-LM-Fc/ΔHep have much strongerpreference for binding to HA as demonstrated in the GAG competitiveassay. As shown in Example 11, at least 10 fold more of chondroitinsulfate A was needed to reach the similar competitive inhibition as HA.In addition, in a separate experiment, biotinylated-HABP was compared tobiotinylated-TSG-6-LM-Fc in a GAG competitive assay, and similarinhibition patterns of four GAG chains (HA, Heparin Chondroitin sulfatesA & C) to the binding of biotin-HABP to HA versus the binding ofTSG-6-LM-Fc to HA were observed.

Example 13 Quantitation of Hyaluronan in K₃-EDTA Human Plasma byAggrecan Binding Assay

The concentration of hyaluronan was determined in clinical human plasmasamples using a sandwich binding assay. Plasma samples were obtainedfrom 19 subjects with solid tumor and various tumor types at advancedstage that were enrolled in a clinical study (Phase 1-101 and Phase1-102; see Table 11)) assessing escalating dosage of PEGPH20 in patientsin the presence or absence of dexamethasone. In addition, plasma samplesalso were obtained from twenty (20) normal patients (obtained fromBioReclamation, Hicksville, N.Y.). Prior to treatment with PEGPH20,baseline levels of HA were determined as follows.

Immulon 4HBX 96-well flat bottom microtiter plates (Immulon/Thermo;Catalog No. 3855) were coated with a recombinant human aggrecan(rHu-aggrecan) R & D Systems, Catalog No. 842162) as capture reagent.Prior to use, the rHu-aggrecan was reconstituted by adding 250 μl ofreagent diluent to 1 vial and stored at 2-8° C. for up to 1 month. Then,to generate a 0.5 μg/mL solution of rHu-aggrecan, a 240-fold dilution ofthe stock was prepared (e.g. 41.7 μL stock to about 10 mL PBS).Immediately after dilution, 100 μL was dispensed into each well of a4HBX plate and the plate was covered with a plate sealer and incubatedovernight or up to 3 days at room temperature. After incubation, eachwell in the plate was washed five (5) times with 1×PBST wash buffer(1×PBS, 0.05% Tween 20) using the ELx405Select CW plate washer. Theassay plate was then blocked with block buffer (5% Tween 20 in PBS) byadding 300 μL of block buffer to each well. The plate was covered withan adhesive plate cover and incubated at ambient temperature for atleast 1 hour without shaking.

Prior to incubating the plate with sample, plasma samples and a standardcurve were prepared. Briefly, plasma test samples were obtained andstored at ≦60° C. until analyses. Immediately prior to analyses, thetest samples were thawed on wet ice and mixed briefly by vortexing justprior to dilution. Then, several serial dilutions of plasma test sampledilutions were prepared in order to ensure at least one sample dilutionfell within the range of the calibration curve by dilution in ReagentDiluent (5% Tween-20 PBS solution, prepared by adding 6.5 mL Tween-20(Sigma; Catalog No. P7949) to 123.5 mL Phosphate Buffered Saline (PBS;CellGro; Catalog No. 21-031-CV)). To assess assay validity, two qualitycontrol samples also were diluted for assay. The controls were pooledhuman plasma collected in K₃-EDTA (pooled human K₃-EDTA plasma; “lowquality control”) and pooled human K₃-EDTA plasma spiked with exogenoushyaluronan (HA) (“high quality control”). The minimum required dilution(MRD) for human K3-EDTA plasma (used as a control) was 1:4. Dilutionswere in polypropylene tubes (e.g., BioRad Titer tubes; BioRad, CatalogNo. 223-9391) and were made to a total volume (sample and diluent) of500 Each dilution was mixed as it was prepared by brief pulse-vortexing.Pipets were changed in between each dilution.

For the standard curve, a hyaluronan stock (132 kD, 1800 ng/mL; R& DSystems, Catalog No. 842164) was diluted by serial dilution in reagentdiluent (5% Tween 20 in PBS) to final concentrations of 500 ng/mL, 167ng/mL, 55.6 ng/mL, 18.5 ng/mL, 6.2 ng/mL, 2.1 ng/mL, and 0.68 ng/mL. Ablank well containing reagent diluent also was included in the standard.

Then, at the end of the block step, each well was washed five (5) timeswith 1×PBST wash buffer (1×PBS, 0.05% Tween 20) using the ELx405SelectCW plate washer. The test samples, controls and standard curve wereadded to the coated and blocked plate by adding 100 μL of each intriplicate to wells of the plate. The plate was covered with an adhesiveplate sealer and incubated at ambient temperature for approximately 2hours. After incubation, each well was washed five (5) times with 1×PBSTwash buffer (1×PBS, 0.05% Tween 20) using the ELx405Select CW platewasher.

To detect binding of HA to the coated rHu-aggrecan, a biotinylatedrHuAggrecan detection reagent (72 μg/mL; R& D Systems, Catalog No.842163) was added to the plate. First, 10 mL of a 0.3 μg/mLbiotinylated-aggrecan solution was made by diluting the stock solution240-fold in reagent diluent (5% Tween/PBS). Then, 100 μL of thedetection reagent was added to each well. The plate was covered with anadhesive seal and incubated at ambient temperature for approximately 2hours. Each well was washed five (5) times with 1×PBST wash buffer(1×PBS, 0.05% Tween 20) using the ELx405Select CW plate washer. Then, anStreptavidin-HRP (SA-HRP; R&D Systems, Catalog No. 890803) workingsolution was prepared in reagent diluent by diluting the stock 200-fold.Then, 100 μL of the dilute SA-HRP working solution was added to eachwell. The plate was covered with an adhesive seal and incubated atambient temperature for approximately 20 minutes with shaking at 500rpm. At the end of the SA-HRP incubation period, each well was washedfive (5) times with 1×PBST wash buffer (1×PBS, 0.05% Tween 20) using theELx405Select CW plate washer. Then, 100 μL of a TMB substrate (KPL;Catalog No. 52-00-03), which was equilibrated to ambient temperatureprotected from light, was added to each well and incubated at ambienttemperature for 20 minutes with shaking at 500 rpm. Then, 100 μL of TMBstop solution (KPL; Catalog No. 50-85-06) was added to each well for atleast 5 minutes but less than 30 minutes prior to determining theoptical density at 450 nm (OD 450 nm) using a microtiter platespectrophotometer and SoftMax Pro software.

Based on the OD450 nm value, the concentration of intact hyaluronan foreach sample was determined by interpolating from the standard curve. Theresults were multiplied by the sample dilution factor. The data wasreported as the average of all values within the limits of quantitationof the calibration curve in ng/mL. The results are set forth in Tables11 and 12. The results show that the median plasma HA in healthy humanswas 0.015 μg/mL while in phase 1 subjects it was 0.06 μg/mL. Thisrepresented a statistically significant difference with a p<0.0001.

TABLE 11 Plasma HA from Subjects with Tumors Result Subject Tumor TypeAge Sex (ng/mL) Trial 101 1 Histiocytoma 86 M 44.1 2 Colorectal 62 M32.8 3 Rectal 60 M 53.2 4 Pancreatic 57 F 59.8 5 Bladder 63 M 20.3 6Colon 66 F 52.2 7 Pancreatic 63 M 19.5 8 Carcinoid 56 M 62.6 9 Ovarian70 F 82.3 10 Colon 60 F 254.6 11 Prostate 78 M 61.2 12 Non small cell 61F 348.3 lung cancer 13 Prostate 71 M 30.4 14 Prostate 55 M 82.4 Trial102 15 Ovarian 55 F 67.3 16 Esophageal 71 M 88.6 17 NSCLC 65 F 59.7 18colon w/liver mets 72 F 55.4 19 colo-rectal 62 F 207.8

TABLE 12 Plasma HA from Healthy Subjects Result Subject Age Sex (ng/mL)1 45 M 15.1 2 44 M 25.4 3 43 M 11.2 4 31 M 18.3 5 47 M 63.2 6 26 F 17 728 F 13.4 8 21 F 13.4 9 41 F 12.8 10 24 F 12.6 11 19 F 7.6 12 33 F 18.413 28 F 18.5 14 21 F 14.5 15 35 F 19 16 54 M 11.7 17 37 M 21.9 18 38 M8.3 19 58 M 37.5 20 49 M 8.6

Example 14 Histochemical Detection of HA

Histochemical detection of HA were obtained from a pre-biopsy tumorspecimen and a post-treatment metastatic liver biopsy sample from apatient dosed for 4 weeks with 1.6 μg/kg PEGPH20+dexamethasone. Thepre-dose biopsy (pre biopsy) was an archived sample obtained in 2007(3.5 years prior to the treatment with PEGPH20). The post-treatmentbiopsy sample was obtained 3 days after the last dose (8^(th) dose) in aPEGPH20 plus dexamethasone treatment regimine from a female colon cancerpatient with liver metastases. Specifically, the patient post-treatmentbiopsy was obtained after one cycle of PEGPH20 treatment at 1.6 μg/kg ona twice weekly schedule for the cycle of administration withdexamethasone co-treatment. The treatment cycle was defined as a 28-dayperiod, with PEGPH20 administered intravenously (IV) and dexamethasoneadministered orally. On each dosing day, a premedication regimen of 4 mgof dexamethasone was administered orally one hour prior to the PEGPH20,followed by a second dose of 4 mg dexamethasone 8-12 hours after PEGPH20dosing.

Briefly, the tumor biopsies were fixed in normal buffered formalin (NBF)and 5 μm sections cut and stained using a biotin labeled hyaluronanbinding protein (HABP-bio) (Seikagaku, Japan). After washing to removethe primary reagent, a labeled secondary reagent was used. Nuclei werecounter-stained using a DAPI (4′,6-diamidino-2-phenylindole) reagent.Micrographs were captured via a Nikon Eclipse TE2000U invertedfluorescent microscope coupled to a Insight FireWire digital camera(Diagnostic Instruments, Michigan) or ZEISS overhead scope (Carl Zeiss,Inc.) that has the same imaging system.

The histochemical staining of the samples with biotinylated-HA bindingprotein demonstrated a decrease in pericellular and stromal HA levelsafter one cycle of PEGPH20 treatment. The results are summarized inTable 13. The H score represents the relative intensity of pericellularand stromal HA. The data demonstrates the ability of PEGPH20 to degradetumor-associated HA as demonstrated by a reduction of HA staining in thetumor biopsy after treatment.

TABLE 13 Histochemical Detection of HA Pericellular tumor Stroma cells(% cells stained) (% area stained) % total area Specimen 3+ 2+ 1+ 0 H 3+2+ 1+ 0 H Tumor Stroma** prebiopsy 10 30 25 35 115 30 50 15 5 205 40 50postbiopsy 0 0 25 75 25 30 30 23 17 173 20 5 **tumor associated stroma

Example 15 HPLC Method for the Estimation of Hyaluronan (HA) Level inPlasma

This Example describes a method for the determination of theHA-disaccharide content in plasma as a measure of HA catabolites, whichare the breakdown products after enzymatic activity of PEGPH20. Themethod employs the hydrolysis of HA with Chondroitinase ABC to releasethe HA-disaccharides, derivatize them with 2-amino acridone (AMAC) andanalyze them on a reverse-phase HPLC coupled with fluorescencedetection. Quantitation of the HA-disaccharides is accomplished bycomparison with HA-disaccharide standards. This assay was used tomeasure the enzymatic activity of PEGPH20 by monitoring concentrationsof hyaluronan catabolites in plasma of patients that were selected atschedules times from patients after treatment with PEGPH20.

1. Working Standards

In the method, a working standard solution was generated. First, adilute stock solution (DSS) was generated from an HA-disaccharide StockSolution (SS). The HA disaccharide SS was generated by adding 1 mL ofwater to a vial of HA-Disac (V-labs, Cat. No. C3209) containing 2 mg oflyophilized powder to make a uniform suspension. To generate dilutestock solutions, 5 μl of the SS solution was diluted with 125 μL ofwater to generate a DSS1 solution (containing 200 pmoles/μl HA-Disac;200 nmoles/ml HA-Disac). Five-fold serial dilutions in water were madeto generate DSS2 (containing 40 pmoles/μl HA-Disac; 40 nmoles/mlHA-Disac) and then DSS3 (containing 8 pmoles/μl HA-Disac; 8 nmoles/mlHA-Disac). Next, working standard solutions were generated as set forthin 25% human serum albumin (HSA) (ABO Pharmaceuticals, Cat. No. 1500233)or Normal Mouse plasma (Bioreclamation, Cat. No. MSEPLEDTA2-BALB-M) asset forth in Tables 14 and 15.

TABLE 14 Working Standard Solution in HSA 25% HA0Disac Water HSA (pmolesWSS# DSS3 DSS2 DSS1 (μl) (μl) in 150 μl) WSS0 0.00 130.00 20.00 0 WSS11.25 128.70 20.00 10 WSS2 3.13 126.87 20.00 25 WSS3 6.25 123.75 20.00 50WSS4 12.50 117.50 20.00 100 WSS5 6.25 123.75 20.00 250 WSS6 12.50 117.5020.00 500 WSS7 6.25 123.75 20.00 1250 WSS8 12.50 117.50 20.00 2500 WSS925.00 105.00 20.00 5000 WSS10 50.00 80.00 20.00 10000

TABLE 15 Working Standard Solution in Normal Mouse Plasma Normal MouseHA0Disac Water Plasma (pmoles WSS# DSS3 DSS2 DSS1 (μl) (μl) in 150 μl)WSS0 0.00 50.00 100.00 0 WSS1 1.25 48.70 100.00 10 WSS2 3.13 46.87100.00 25 WSS3 6.25 43.75 100.00 50 WSS4 12.50 37.50 100.00 100 WSS56.25 43.75 100.00 250 WSS6 12.50 37.50 100.00 500 WSS7 6.25 43.75 100.001250 WSS8 12.50 37.50 100.00 2500 WSS9 25.00 25.00 100.00 5000 WSS1050.00 00.00 100.00 10000

2. Hydrolysis and Derivation of Samples

Next, the sample was hydrolyzed. The sample (e.g. plasma) was preparedby taking approximately 100 μg of protein in a polypropylene tube andadjusting the volume to 340 μl with water. A matrix blank also wasprepared by taking dilution buffer (1.59 g HEPES, 5.07 g NaCl, 1800 mLwater, pH 7.0) equal to the volume of the sample and adjusting thevolume to 340 Hydrolysis of the samples and matrix blank were effectedby adding 60 μl of TFA to the sample tube and matrix blank tube and thecontents were mixed and incubated at 100° C. for 4 hours. The vials wereallowed to cool to room temperature. The vials were evaporated todryness using a speed vac. Then, 300 μl of water was added to each tubeand vortexed to resuspend the samples.

For derivation of hydrolyzed samples, blanks and working samples, 45 μlof each sample (sample, blank or working sample) was evaporated todryness in a speed vac. Then, 10 μl of SAS was added to the driedsample, blank and working standards. Then, 50 μl ABA/NaCNBH3 labelingsolution was added. The tubes were vortexed and centrifuged briefly.Then, 440 μl of Mobile Phase A was added and the tubes were mixed well.Mobile Phase A was prepared as follows: 132 mL of 1 M ammonium acetatebuffer (Sigma, Cat. No. A7330) was added to a 1 L volumetric flask andwater added to fill the flask. Following derivation, nominal on-columnloads per 20 μL of injection for the working standards is as set forthin Table 16.

TABLE 16 Fuc GalN GlcN Gal Man WSS# (pmol) (pmol) (pmol) (pmol) (pmol)WSS1 25 3.0 105 42 175 WSS2 30 3.7 127 51 211 WSS3 35 4.3 150 60 250WSS4 41 5.0 173 69 287 WSS5 46 5.6 195 78 325

3. HPLC

The HPLC column was equilibrated at a flow rate of 1.0 mL/min with theinitial mobile phase settings as outlined in Table 17. The system wasallowed to equilibrate until the baseline was steady. HPLC analysis wasperformed with the instrument parameters as outlined in Table 17.

TABLE 17 HPLC Instrument Parameters Parameter Values Column BakerbondC18 reversed phase column, 4.6 × 250 mm, 5 um Column Temperature RoomTemperature Mobile Phase A 0.2% n-butylamine, 0.5% phosphoric acid, 1%tetrahydrofuran in water Mobile Phase B 50% mobile phase A, 50%acetonitrile Flow Rate 1.0 mL/min Injection volume 20 μl DetectorFluorescence; Excitation 360 nm, Emission 425 nm Sample condition 4-6°C. Gradient Time (min) % A % B 0.0 95 5 25.0 95 5 50.0 85 15 50.1 0 10060.0 0 100 60.1 95 5 70.0 95 5

The sequence for sample analysis was as follows: WSS5 (1 injection) forcolumn conditioning/equilibration/detector gain; water injection (1injection); WSS3 (3 injections); WSS1 (1 injection); WSS2 (1 injection);WSS4 (1 injection); WSS5 (1 injection); Water (1 injection); MatrixBlank (1 injection); Sample 1 (1 injection); Sample 2 (1 injection);WSS3 (3 injections); Water (1 injection). The system was consideredsuitable when there was acceptable separation quality; the signal tonoise ratio for the shorter monosaccharide peak in the WSS1 sample wasequal to or more than 10; the relative standard deviation (RSD) of thepeak areas for each monosaccharide standard for the 6 injections of WSS3was equal or less than 4%; the correlation coefficient (r) was 0.99 (rwas measured using software to plot the peak area of each workingstandard against the on-column load (expressed as pmol) using the firstthree injections of the WSS3 standard and calculating the slope,intercept and correlation coefficient for the working standards using alinear least square regression model); the peak areas for peakscorresponding to monosaccharides were no more than 2% of the peak areameasured for WSS5; and the peak areas for peaks corresponding tomonosaccharides in water injection were no more than 0.5% of the peakareas measured for WSS5.

4. Sample Analysis

The average corrected peak area for each monosaccharide in each samplepreparation was determined. Valley-to-valley integration was used forthe GalN peak. To determine this, the linear curves generated from theworking standards were used to calculate the amount of eachmonosaccharide loaded for each sample preparation. For each type ofmonosaccharide, the average molar ratio of monosaccharides per proteinmolecule for each sample was calculated. Then, for each sample, theoverall sum of the average molar ratios for all five monosaccharides wasdetermined. The calculations were performed based on the following:Molecular weight (MW) of non-glycosylated hyaluronidase protein is 51106g/mol; the total volume of each sample was 500 μL; the sample dilutionfactor is 0.15; the volume of each injection is 20 μL; and theconversion factor from mg to pg is 10⁹. The calculations were performedas follows for each monosaccharide:

The amount of monosaccharide for each preparation was calculated usingthe following equation:

${{Monosaccharide}({pmol})} = \frac{{{Peak}\mspace{14mu} {Area}} - {Intercept}}{Slope}$

The number of monosaccharides per protein molecule was calculated byusing the following formula:

${{Monosaccharides}\mspace{14mu} {per}\mspace{14mu} {protein}\mspace{14mu} {ratio}} = \frac{{{monosaccharide}({pmol})} \times {MW} \times 500\mspace{14mu} \mu \; 1}{0.1\mspace{14mu} {mg} \times 10^{9} \times 20\mspace{14mu} \mu \; 1 \times 0.15}$

For each sample, the results for each sample were reported as themonosaccharides per protein ratio for each monosaccharide along with thesum of the five monosaccharide ratios.

5. Results

The disaccharide assay described above was used to measure HA and itscatabolites from patients enrolled in phase I clinical studies thatreceived IV doses of PEGPH20 at doses that ranged from 0.5 μg/kg to 50μg/kg over a dosage regime cycle with or without dexamethasone. PlasmaHA concentrations prior to PEGPH20 dosing were typically less than 1μg/mL or below the level of quantification (0.5 μg/mL) for all patientsin the study.

Plasma collected from a patient that received a single 50 μg/kg dose ofPEGPH20 was assessed over time after treatment. The results show thatplasma concentrations of hyaluronan increased significantly. In thispatient, while PEGPH20 concentrations declined with a terminal half-lifeof 2 days, elevated concentrations of HA catabolites accumulated moreslowly and persist for up to 2 weeks post-PEGPH20 treatment with amaximum HA plasma concentration observed about 200 hours post-PEGPH20dose.

Plasma also was collected from 12 additional patients beginning 24-hourspost initial PEGPH20 dose that were either treated with 0.5 μg/kgPEGPH20 twice weekly (1 patient), 0.5 μg/kg every 21 days (3 patients),0.75 μg/kg every 21 days (4 patients), 1.0 μg/kg every 21 days (3patients), or 1.5 μg/kg every 21 days (1 patients). The results showthat following single or multiple doses of PEGPH20 that ranged from 0.5μg/kg to 1.5 μg/kg, HA catabolite levels increased in a dose-dependentmanner over the course of a week. Maximal HA concentrations (C_(max))and one-week area-under-the-curve-estimates (AUC_(0-168h)) were alsodetermined for each patient to quantify the pharmacodynamic response.The results showed that systemic exposure to HA catabolites, as measuredby maximum plasma concentration or area-under-the-curve increased withincreasing dose of PEGPH20. In addition, blood samples from patientsadministered with PEGPH20 where dexamethasone was added to a dosingregime as a premedication to eliminate or ameliorate the musculoskeletaleffects caused by PEGPH20 administration. The treatment cycle wasdefined as a 28-day period, with PEGPH20 administered intravenously (IV)and dexamethasone administered orally. Dosing of PEGPH20 anddexamethasone took place on days 1, 4, 8, 11, 15, 18, 22 and 25 of the28-day cycle. On each dosing day, a premedication regimen of 4 mg ofdexamethasone was administered orally one hour prior to the PEGPH20,followed by a second dose of 4 mg dexamethasone 8-12 hours after PEGPH20dosing. Plasma was taken at various time points after administration ofPEGPH20 during the first week of treatment. Consistent with theobservations in the samples from patients receiving only PH20 describedabove, plasma HA concentration vs. time data increased afteradministration of PEGPH20. Concentrations of plasma HA measured duringthe first week of dosing increased with increasing dose of PEGPH20. Inthree patients that completed a full cycle of treatment and received 8doses of PEGPH20, the results showed a sustained increased plasma HAconcentrations in samples from all three patients measured throughoutthe dosing period.

These results are consistent with the expected mechanism of PEGPH20activity and support the role of HA as a biomarker for PEGPH20pharmacodynamics.

Example 16 Magnetic Resonance Imaging

Diffusion weighted MRI was performed using a single shot spin-echosequence to estimate pixel-by-pixel values for apparent diffusioncoefficient. Dynamic contrast enhanced magnetic resonance imaging(DCE-MRI) included imaging during infusion with a contrast agent.Calibration was accomplished using a two part phantom containing aninner tube and ice/water mixture. Scans were performed pre-treatment andpost-treatment.

1. Apparent Diffusion Coefficient Magnetic Resonance Imaging (ADC-MRI)

Apparent diffusion coefficient magnetic Resonance imaging (ADC-MRI)measures the volume of water that has moved across the cell membranebased upon a calculation derived from the pre- and post-treatment scans.ADC-MRI scans were completed for a total of 10 of the 14 patients in aphase I clinical study assessing PEGPH20 treatment without dexamethasonepremedication and in 4 of the 5 patients in a phase I clinical studyassessing PEGPH20 treatment with dexamethasone premedication. Analysisof the images acquired from each patient was performed by a radiologistat Imaging Endpoints (Scottsdale, Ariz.), and quantitative estimates ofADC were computed for tissues in each patient. A summary of the ADC-MRIfindings associated with tumor regions is shown in Table 18. As shown inthe Table, increases in ADC-MRI were observed in 7 of 14 (50%) ofpatients following PEGPH20 dosing. Increased ADC values are consistentwith the mechanism of action of PEGPH20. ADC values, however, did notchange in 5 of 14 patients, and values decreased in 2 of 14 patients.

TABLE 18 ADC-MRI Summary Post-Dose Change in Tumor ADC- Dose & FrequencyScan Days MRI from baseline Example 15 50 μg/kg D 4 no change 0.5 μg/kg;2x/wk D 3 no change 0.5 μg/kg; 21 day cycle D 3 increase 0.5 μg/kg; 21day cycle D 4 decrease in lymph nodes 0.5 μg/kg; 21 day cycle D 3increase 0.75 μg/kg; 21 day cycle D 3 increase 0.75 μg/kg; 21 day cycleD 3 no change 0.75 μg/kg; 21 day cycle D 3, D 30 increase 1.0 μg/kg; 21day cycle D 5 increase 1.5 μg/kg; 21 day cycle D 3 no change Example 141.6 μg/kg + D 3, D 29 increase dexamethasone; 2x/wk 5.0 μg/kg + D 1, D 4increase D 1 dexamethasone; 2x/wk 1.6 μg/kg + D 2, D 25 decrease D 25dexamethasone; 2x/wk 1.6 μg/kg + D 1, D 2 no change dexamethasone; 2x/wk

2. Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI)

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) measuresblood flow that indicates a change in tumor's vascularity. Scans werecompleted in 4 patients in a phase I clinical study assessing PEGPH20treatment with dexamethasone premedication. Analysis of images acquiredfrom each patient was performed by a radiologist at Imaging Endpoints(Scottsdale, Ariz.), and quantitative estimates of volume transfercoefficient (Ktrans), blood volume (Vp) and extracellular volumefraction (Ve) were computed for tissues in each patient. A summary ofthe DCE-MRI findings associated with tumor regions is set forth in Table19. Significant increases in the Ktrans parameter were observed in thetwo patients that were scanned on the day of PEGPH20 dosing. Theincrease in Ktrans within hours of dosing is consistent with preclinicaldata that show PEGPH20 causes vascular decompression and increased bloodflow (Thompson et al. (2010) Mol. Cancer Ther., 9:3052-64).

TABLE 19 DCE-MRI Summary Post-Dose Change in Tumor DCE- Dose & FrequencyScan Days MRI from baseline 1.6 μg/kg + D 3, D 29 decrease in ktrans atD29 dexamethasone; 2x/wk 5.0 μg/kg + D 1, D 4 increase in ktrans, Ve, Vpdexamethasone; 2x/wk (8 hr). Return to baseline (D 4) 1.6 μg/kg + D 2, D25 No baseline scan available. dexamethasone; 2x/wk Increase in Vp on D25 vs. D 2. No change in Ktrans 1.6 μg/kg + D 1, D 2 Increase in Ktrans(8 hr, dexamethasone; 2x/wk 24 hr. Increase in Ve for lung tumor but notliver tumor (D 1, D 2). No change in Vp

DCE-MRI imaging also was performed on a further patient with apancreatic tumor enrolled in a phase I clinical study receiving 3.0μg/kg+dexamethasone/wk in a cycle of administration for 28 days. Imagingwas performed pre-dose and post-dose as follows: 8 hours (Day 1), 24hours (Day 2) and 3 days post the 4^(th) weekly PEGPH20 dose in cycle 1(end of cycle 1). The results are set forth in Table 20. The resultsshows that PEGPH20 increases tumor Ktrans measured by serial DCE-MRI.

TABLE 20 DCE-MRI Results from Patient Dosed 3.0 μg/kg + dexamethasone/wkBaseline Day 1 Day 2 End of Cycle 1 Mean K^(trans) 0.057 0.147 0.2420.212

3. FDG-PET Imaging

Positron emission tomography (PET) using FDG, an analogue of glucose,was used to give tissue metabolic activity in terms of regional glucoseuptake. The FDG-PET imaging was performed on a patient with metastaticrectal carcinoma with lung metastasis enrolled in a phase I clinicalstudy receiving 3.0 μg/kg+dexamethasone; 2×/wk in a cycle ofadministration for 28 days. Imaging was performed pre-dose, 8 hourspost-dose, 24 hours post-dose and at the end of the cycle (1 day afterthe 8^(th) dose). The FDG standardized uptake value (SUV) was determinedusing standard methods. The results are set forth in Table 21. Theresults showed that the patient exhibited decreased tumor metabolicactivity post-PEGPH20 treatment of landmark pulmonary metastases.

TABLE 21 FDG-PET Results From Patient Dosed 3.0 μg/kg + dexamethasone;2x/wk Δ Δ Δ 24 h baseline Anatomical Baseline 8 h baseline 24 h Δ 8 hDay 26 to Day to Day Site (SUV) (SUV) to 8 h (SUV) to 24 h (SUV) 26 26Superior 12.9 9.4 −27% 8 −15% 8 0% −38% segment of left lower lobe leftlung 11.2 9.1 −19% 7.1 −22% 6.7 −6% −40% base right upper 6.8 4.5 −34%3.9 −13% 4 +3% −41% lobe at right perihilar region right lower 8.1 5.4−33% 5.2 −4% 4.7 −10% −42% lobe

4. Summary

The results show that various tumor imaging modalities can be used todemonstrate and monitor activity of PEGPH20 in tumor tissue.

Example 17 TSG-6-Fc Tumor-Targeted Imaging for HA-Rich Cancer Diagnosisand Treatment

Hyaluronan-rich tumor-bearing mice or control mice were administeredwith TSG-6-LM-Fc/ΔHep labeled with DyLight 755 Fluor Labeling reagent(TSG-6-LM-Fc/ΔHep^(DL755)), and mice were imaged to assess tumor-bindingand distribution of TSG-6-LM-Fc/ΔHep^(DL755). Specificity also wasassessed by comparing staining and distribution to an IgG^(DL755)control. For generation of BxPC3 peritibial tumor-bearing mice, micewere inoculated with BxPC-3 human pancreatic adenocarcinoma (ATCCCRL-1687) tumor cells subcutaneously (s.c., right hind leg) at 1×10⁷cells/0.1 mL. For generation of HA⁺³ Du145-Has2 and HA-Du145tumor-bearing mice, mice were inoculated with both Du145-Has2 cells(generated as described above) and Du145 cells peritibially(intramuscular injection adjacent to the right tibia periosteum oneither side) at 5×10⁶/0.05 mL

TSG-6-LM-Fc/ΔHep^(DL755) was generated by fluorescently labelingTSG-6-LM-Fc/ΔHep (generated as described in Example 9) with DyLight 755using the Thermo Scientific DyLight 755 Amine-Reactive Dye kit (CatalogNo. 84538; Thermo Scientific, Rockford, Ill.) according to themanufacturers protocol.

A. Distribution of TSG-6-LM-Fc/ΔHep^(DL755) with and withoutPretreatment with PEGPH20

Mice bearing an HA⁺² BxPC3 peritibial tumor at about 18-20 mm indiameter were injected intravenously with 1 μg, 5 μg or 10 μgTSG-6-LM-Fc/ΔHep^(DL755). In one group of mice, mice were pretreatedwith intravenous administration of PEGPH20 at 4.5 mg/kg three (3) hoursprior to administration of TSG-6-LM-Fc/ΔHep^(DL755).

A fluorescent whole body image system (IVIS Lumina XR, Caliper LifeSciences, Mountain View, Calif.) was used to track fluorescence in theanimal. Selective excitation of DyLight755 was done using a D745 nmband-pass filter, and the emitted fluorescence was collected through along-pass D800 nm filter. The 3 groups of mice (non-injected,TSG-6-LM-Fc/ΔHep^(DL755), and PEGPH20+TSG-6-LM-Fc/ΔHep^(DL755)) wereimaged at various timepoints post TSG-6-LM-Fc/ΔHep^(DL755) (1 hours, 4hours, day 1, day 2, day 3, day 4, day 5 and day 6). For imaging,non-injected control mice also were assessed. Fluorescent images werecaptured with a super cooled, high sensitivity, digital camera.Fluorescent images were later analyzed with Living Image (Caliper LifeSciences, Mountain View, Calif.).

The results show that by 1 hour and 4 hours after injection,TSG-6-LM-Fc/ΔHep^(DL755) was detected as circulating in the bloodstream, and also was detected as starting to bind to the tumor. Thebinding to the tumor was dose-dependent, with increased stainingintensity observed with the 10 μg dose. Less tumor binding was detectedby imaging in mice treated with PEGPH20 at all doses and time points. Atlater time points after injection (e.g. day 1 or day 2), liver bindingalso was detected, although this was less in the mice injected with the1 μg low dose of TSG-6-LM-Fc/ΔHep^(DL755). TSG-6-LM-Fc/ΔHep^(DL755)reached peak levels between day 1 and 2 as assessed by image analysis.In low-dose treated mice, TSG-6-LM-Fc/ΔHep^(DL755) was eliminated day 3after injection. TSG-6-LM-Fc/ΔHep^(DL755) was sill circulating inhigh-dose treated mice 5 days post injection, and all binding to thetumor was diminished 6 days after injection.

In sum, the in vivo imaging results show that TSG-6-LM-Fc/ΔHep^(DL755)binding was dose-dependent and reached peaked levels 1-2 dayspost-injection. Further, HA removal by PEGPH20 resulted in lessTSG-6-LM-Fc/ΔHep^(DL755) binding. TSG-6-LM-Fc/ΔHep^(DL755) binding waseliminated from the tumor 6 days post injection.

B. Comparison of TSG-6-LM-Fc/ΔHep^(DL755) Binding Between Du145Tumor+/−Has2

HA⁺³ Du145-Has2 and HA-Du145 tumor-bearing mice were injectedintravenously with 5 μg TSG-6-LM-Fc/ΔHep^(DL755). The mice were imageddaily post TSG-6-LM-Fc/ΔHep^(DL755) injection. Although a low-levelbackground staining of HA-DU145 tumor was detected, there was much moreTSG-6-LM-Fc/ΔHep^(DL755) binding to HA-rich Du145-Has2 as assessed byimage results. The binding peaked at day 1-2 as determined by stainingintensity. Thus, the results show that the more HA that is present inthe tumor, the more TSG-6-LM-Fc/ΔHep^(DL755) binds to the tumor.

C. Targeting Specificity of TSG-6-LM-Fc/ΔHep^(DL755).

The specificity of TSG-6-LM-Fc/ΔHep^(DL755) for HA-rich tumors wasfurther assessed by comparing binding of TSG-6-LM-Fc/ΔHep^(DL755) orIgG^(DL755) to HA⁺² BxPC3 peritibial tumor-bearing mice. HA⁺²BxPC3peritibial tumor-bearing mice were injected intravenously with 5 μgTSG-6-LM-Fc/ΔHep^(DL755) or with 5 μg IgG^(DL755). The mice were imageddaily after injection. The imaging results showed little to nodetectable staining of IgG^(DL755) to the tumor, and thus greaterbinding of TSG-6-LM-Fc/ΔHep^(DL755) to PC3 tumor than IgG^(DL755).

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A method for treating a tumor in a subject with an anti-hyaluronanagent, comprising: a) contacting a tissue or body fluid sample from asubject with a tumor or cancer with a hyaluronan binding protein (HABP)molecule, wherein the HABP has not been prepared from or isolated fromanimal cartilage; b) detecting binding of the hyaluronan binding proteinto the sample, thereby determining the amount of hyaluronan in thesample, wherein if the amount of hyaluronan in the sample is at or abovea predetermined threshold level, selecting the subject for treatmentwith an anti-hyaluronan agent; and c) administering an anti-hyaluronanagent to the selected subject, thereby treating the selected subjectwith an anti-hyaluronan agent.
 2. The method of claim 1, wherein thepredetermined threshold level is high HA.
 3. The method of claim 1,wherein: the HABP molecule comprises a link module; or the HABP moleculecomprises a G1 domain of a type C hyaluronan binding protein.
 4. Themethod of claim 3, wherein the link module or G1 domain is the only HABPportion of the molecule.
 5. The method of claim 3, wherein: the HABPmolecule comprises a link module and the link module is selected fromamong CD44, LYVE-1, HAPLN1/link protein, HAPLN2, HAPLN3, HAPLN4,aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, Stabilin-1,Stabilin-2, CAB61358 and KIA0527, a portion thereof comprising the linkmodule and a sufficient portion of a link module to bind HA; or the HABPcomprises a G1 domain and the G1 domain is selected from among AggrecanG1, Versican G1, Neurocan G1 and Brevican G1.
 6. The method of claim 1,wherein the HABP is a multimer comprising a first HA-binding domainlinked directly or indirectly via a linker to a multimerization domainand a second HA-binding domain linked directly or indirectly via alinker to a multimerization domain.
 7. The method of claim 6, whereinthe multimerization domain is selected from among an immunoglobulinconstant region (Fc), a leucine zipper, complementary hydrophobicregions, complementary hydrophilic regions, compatible protein-proteininteraction domains, free thiols that form an intermolecular disulfidebond between two molecules, and a protuberance-into-cavity and acompensatory cavity of identical or similar size that form stablemultimers, or a variant thereof that effects multimerization.
 8. Themethod of claim 1, wherein the HABP has a dissociation constant (K_(d))of less than 1×10⁻⁷ M.
 9. The method of claim 1, wherein thehyaluronan-associated disease or condition is a tumor or cancer.
 10. Themethod of claim 1, wherein the anti-hyaluronan agent is a hyaluronandegrading enzyme that is a hyaluronidase.
 11. The method of claim 10,wherein the hyaluronan-degrading enzyme is a PH20 hyaluronidase or atruncated form thereof lacking a C-terminal glycosylphosphatidylinositol(GPI) attachment site or a portion of the GPI attachment site, wherebythe truncated form exhibits hyaluronidase activity.
 12. The method ofclaim 1, wherein the anti-hyaluronan agent is a hyaluronan-degradingenzyme that is modified by conjugation to a polymer.
 13. The method ofclaim 12, wherein the polymer is polyethylene glycol (PEG) and thehyaluronan degrading enzyme is PEGylated.
 14. A method for treating asubject with a tumor, comprising: a) administering an anti-hyaluronanagent to a subject; b) contacting a tissue or body fluid sample from thesubject treated with the anti-hyaluronan agent with a hyaluronan bindingprotein (HABP), wherein the HABP has not been prepared from or isolatedfrom animal cartilage; c) detecting binding of the hyaluronan bindingprotein to the sample, thereby determining the amount of hyaluronan inthe sample; d) comparing the level of hyaluronan to a control orreference sample, thereby determining the amount of hyaluronan in thesample relative to the control or reference sample, wherein detection ofa decrease in hyaluronan compared to the control or reference sampleindicates that the treatment is effective; and e) altering treatmentbased on the determined amount of hyaluronan in the sample relative tothe control or reference sample, wherein: if the amount of hyaluronan inthe sample is at or above the amount in the control or reference sample,continuing treatment or escalating treatment by increasing the dosageand/or dose schedule of the anti-hyaluronan agent; or if the amount ofhyaluronan in the sample is below the amount in the control or referencesample, continuing the treatment, reducing treatment by decreasing thedosage and/or dose schedule or terminating treatment of theanti-hyaluronan agent.
 15. The method of claim 14, wherein the controlor reference sample is a corresponding sample from the subject beforetreatment with the anti-hyaluronan agent or is a corresponding samplefrom the subject after the previous dose of anti-hyaluronan agent. 16.The method of claim 14, wherein the HABP comprises a link module or a G1domain of a type C hyaluronan binding protein.
 17. The method of claim16, wherein the link module or a module of the G1 domain is the onlyHABP portion of the molecule.
 18. The method of claim 14, wherein theHABP is a multimer comprising a first HA-binding domain linked directlyor indirectly via a linker to a multimerization domain and a secondHA-binding domain linked directly or indirectly via a linker to amultimerization domain.
 19. The method of claim 14, wherein the HABP hasa dissociation constant (K_(d)) of less than 1×10⁻⁷ M.
 20. A nucleicacid molecule encoding a TSG-6 multimer, wherein: the nucleic acidmolecule encodes a fusion polypeptide containing a TSG-6 link modulelinked directly or indirectly via a linker to a multimerization domain;and the multimerization domain is a polypeptide that interacts withitself to form a stable protein-protein interaction, whereby the encodedprotein forms a multimer containing at least two TSG-6 link modules. 21.The nucleic acid molecule of claim 20, wherein the link module is theonly TSG-6 portion of the fusion polypeptide.
 22. The nucleic acidmolecule of claim 20, wherein the encoded TSG-link module comprises thesequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 418 ora sequence of amino acids comprising at least 85% amino acid sequenceidentity to the sequence of amino acids set forth in SEQ ID NO: 207,360, 417 or 418 that specifically binds HA.
 23. The nucleic acidmolecule of claim 20, wherein the encoded TSG-6 link module is modifiedto reduce or eliminate binding to heparin.
 24. The nucleic acid moleculeof claim 23, wherein the encoded TSG-6 link module comprises an aminoacid replacement at an amino acid position corresponding to amino acidresidue 20, 34, 41, 54, 56, 72 or 84 set forth in SEQ ID NO:360, wherebya corresponding amino acid residue is identified by alignment to aTSG-6-LM set forth in SEQ ID NO:360.
 25. The nucleic acid molecule ofclaim 24, wherein the amino acid replacement is to a non-basic aminoacid residue selected from among Asp (D), Glu (E), Ser (S), Thr (T), Asn(N), Gln (Q), Ala (A), Val (V), Ile (I), Leu (L), Met (M), Phe (F), Tyr(Y) and Trp (W).
 26. The nucleic acid molecule of claim 25, wherein theencoded TSG-6 link module comprises an amino acid replacementcorresponding to amino acid replacement selected from among K20A, K34Aand K41A in a TSG-6-LM set forth in SEQ ID NO:360 or the replacement atthe corresponding residue in another TSG-6-LM.
 27. The nucleic acidmolecule of claim 26, wherein the encoded TSG-6 link module comprisesthe sequence of amino acids set forth in SEQ ID NO:361 or 416 or asequence of amino acids comprising at least 85% amino acid sequenceidentity to the sequence of amino acids set forth in SEQ ID NO: 361 or416 that specifically binds HA.
 28. The nucleic acid molecule of claim20, wherein the encoded multimerization domain is selected from among animmunoglobulin constant region (Fc), a leucine zipper, complementaryhydrophobic regions, complementary hydrophilic regions, compatibleprotein-protein interaction domains, free thiols that form anintermolecular disulfide bond between two molecules, and aprotuberance-into-cavity and a compensatory cavity of identical orsimilar size that form stable multimers.
 29. The nucleic acid moleculeof claim 28, wherein the encoded multimerization domain is an Fc domainor a variant thereof that effects multimerization.
 30. The nucleic acidmolecule of claim 29, wherein the encoded TSG-6 multimer comprises thesequence of amino acids set forth in SEQ ID NO:212 or 215 or a sequenceof amino acids that exhibits at least 85% amino acid sequence identityto amino acids set forth in SEQ ID NO:212 or 215 and specifically bindsHA.
 31. The nucleic acid molecule of claim 29, comprising the sequenceof nucleotides set forth in SEQ ID NO:211, 214 or 217, or a sequence ofnucleotides that exhibits at least 85% sequence identity to the sequenceof nucleotides set forth in SEQ ID NO: 211, 214 or
 217. 32. The nucleicacid molecule of claim 29, consisting of the sequence of nucleotides setforth in SEQ ID NO:211, 214 or 217, or a sequence of nucleotides thatexhibits at least 85% sequence identity to the sequence of nucleotidesset forth in SEQ ID NO: 211, 214 or
 217. 33. A cell, comprising thenucleic acid molecule of claim
 20. 34. A vector, comprising the nucleicacid molecule of claim
 20. 35. A method of producing a TSG-6 multimer,comprising: introducing the nucleic acid molecule of claim 20 into acell; culturing the cell under conditions whereby the fusion polypeptideis expressed by the cell; and recovering the TSG-6 multimer.